Synthesis of epothilones, intermediates thereto and analogues thereof

ABSTRACT

The present invention provides convergent processes for preparing epothilone A and B, desoxyepothilones A and B, and analogues thereof, useful in the treatment of cancer and cancer which has developed a multidrug-resistant phenotype. Also provided are intermediates useful for preparing said epothilones.

[0001] This application is based on U.S. Provisional Applications SerialNos. 60/075,947, 60/092,319, and 60/097,733, filed Feb. 25, 1998, Jul.9, 1998, and Aug. 24, 1998, respectively, the contents of which arehereby incorporated by reference into this application, and is acontinuation-in-part of U.S. Ser. No. 08/986,025, filed Dec. 3, 1997,which was based on U.S. Provisional Applications Serial Nos. 60/032,282,60/033,767, 60/047,566, 60/047,941, and 60/055,533, filed Dec. 3, 1996,Jan. 14, 1997, May 22, 1997, May 29, 1997, and Aug. 13, 1997,respectively, the contents of which are hereby incorporated by referenceinto this application.

[0002] This invention was made with government support under grantsCA-28824, CA-39821, CA-GM 72231, GM-18248, CA-62948, and A10-9355 fromthe National Institutes of Health, and grant CHE-9504805 from theNational Science Foundation.

FIELD OF THE INVENTION

[0003] The present invention is in the field of epothilone macrolides.In particular, the present invention relates to processes for thepreparation of epothilones A and B, desoxyepothilones A and B, andanalogues thereof which are useful as highly specific, non-toxicanticancer therapeutics. In addition, the invention provides methods ofinhibiting multidrug resistant cells. The present invention alsoprovides novel compositions of matter which serve as intermediates forpreparing the epothilones.

[0004] Throughout this application, various publications are referredto, each of which is hereby incorporated by reference in its entiretyinto this application to more fully describe the state of the art towhich the invention pertains.

BACKGROUND OF THE INVENTION

[0005] Epothilones A and B are highly active anticancer compoundsisolated from the Myxobacteria of the genus Sorangium. The fullstructures of these compounds, arising from an x-ray crystallographicanalysis were determined by Höfle. G. Höfle et al., Angew. Chem. Int.Ed. Engl., 1996, 35, 1567. The total synthesis of the epothilones is animportant goal for several reasons. Taxol® is already a useful resourcein chemotherapy against ovarian and breast cancer and its range ofclinical applicability is expanding. G. I. Georg et al., TaxaneAnticancer Agents; American Cancer Society: San Diego, 1995. Themechanism of the cytotoxic action of Taxol®, at least at the in vitrolevel, involves stabilization of microtubule assemblies. P. B. Schiff etal., Nature (London), 1979, 277, 665. A series of complementary in vitroinvestigations with the epothilones indicated that they share themechanistic theme of the taxoids, possibly down to the binding sites totheir protein target. D. M. Bollag et al., Cancer Res., 1995, 55, 2325.Moreover, the epothilones surpass Taxol® in terms of cytotoxicity andfar surpass it in terms of in vitro efficacy against drug resistantcells. Since multiple drug resistance (MDR) is one of the seriouslimitations of Taxol® (L. M. Landino and T. L. MacDonald in TheChemistry and Pharmacology of Taxol® and its Derivatives, V. Farin, Ed.,Elsevier: New York, 1995, ch. 7, p. 301), any agent which promisesrelief from this problem merits serious attention. Furthermore,formulating the epothilones for clinical use is more straightforwardthan Taxol®.

[0006] Accordingly, the present inventors undertook the total synthesisof the epothilones, and as a result, have developed efficient processesfor synthesizing epothilones A and B, the correspondingdesoxyepothilones, as well as analogues thereof. The present inventionalso provides novel intermediates useful in the synthesis of epothilonesA and B and analogues thereof, compositions derived from suchepothilones and analogues, purified compounds of epothilones A and B,and desoxyepothilones A and B, in addition to methods of use of theepothilone analogues in the treatment of cancer. Unexpectedly, certainepothilones have been found to be effective not only in reversingmulti-drug resistance in cancer cells, both in vitro and in vivo, buthave been determined to be active as collateral sensitive agents, whichare more cytotoxic towards MDR cells than normal cells, and assynergistic agents, which are more active in combination with othercytotoxic agents, such as vinblastin, than the individual drugs would bealone at the same concentrations. Remarkably, the desoxyepothilones ofthe invention have exceptionally high specificity as tumor cytotoxicagents in vivo, more effective and less toxic to normal cells than theprincipal chemotherapeutics currently in use, including Taxol®,vinblastin, adriamycin and camptothecin.

SUMMARY OF THE INVENTION

[0007] One object of the present invention is to provide processes forthe preparation of epothilones A and B, and desoxyepothilones A and B,and related compounds useful as anticancer therapeutics. Another objectof the present invention is to provide various compounds useful asintermediates in the preparation of epothilones A and B as well asanalogues thereof.

[0008] A further object of the present invention is to provide syntheticmethods for preparing such intermediates. An additional object of theinvention is to provide compositions useful in the treatment of subjectssuffering from cancer comprising any of the analogues of the epothilonesavailable through the preparative methods of the invention optionally incombination with pharmaceutical carriers.

[0009] A further object of the invention is to provide methods oftreating subjects suffering from cancer using any of the analogues ofthe epothilones available through the preparative methods of theinvention optionally in combination with pharmaceutical carriers.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1(A) shows a retrosynthetic analysis for epothilone A and B.

[0011]FIG. 1(B) provides synthesis of compound 11. (a) t-BuMe₂OTf,2,6-lutidine, CH₂Cl₂, 98%; (b) (1) DDQ, CH₂Cl₂/H₂O, 89%; (2) (COCl)₂,DMSO, CH₂Cl₂, −78° C.; then Et₃N, −78C→rt, 90%; (c) MeOCH₂PPh₃Cl,t-BuOK, THF, 0° C.→rt, 86%; (d) (1) p-TsOH, dioxane/H₂O, 50° C., 99%;(2) CH₃PPh₃Br, NaHMDS, PhCH₃, 0° C.→rt, 76%; (e) Phl(OCOCF₃)₂, MeOH/THF,rt, 0.25 h, 92%.

[0012]FIG. 2 provides key intermediates in the preparation of 12,13-E-and —Z-deoxyepothilones.

[0013]FIG. 3(A) provides syntheses of key iodinated intermediates usedto prepare hydroxymethylene- and hydroxypropylene-substituted epothilonederivatives.

[0014]FIG. 3(B) provides methods of preparing hydroxymethylene- andhydroxypropylene-substituted epothilone derivatives, said methods beinguseful generally to prepare 12,13-E epothilones wherein R is methyl,ethyl, n-propyl, and n-hexyl from the corresponding E-vinyl iodides.

[0015]FIG. 3(C) shows reactions leading to benzoylatedhydroxymethyl-substituted desoxyepothilone andhydroxymethylene-substituted epothilone (epoxide).

[0016]FIG. 4(A) provides synthesis of compound 19. (a) DHP, PPTS,CH₂Cl₂, rt: (b) (1) Me₃SiCCLi, BF₃.OEt₂, THF, −78° C.; (2) MOMCl,I-Pr₂NEt, Cl(CH₂)₂Cl, 55° C.; (3) PPTiS, MeOH, rt; (c) (1) (COCl)₂,DMSO, CH₂Cl₂, −78° C.; then Et₃N, −78° C.→rt; (2) MeMgBr, Et₂O, 0°C.→rt, (3) TPAP, NMO, 4 Å mol. sieves, CH₂Cl₂, 0° C.→rt; (d) 16, n-BuLi,THF, −78° C.; then 15, THF, −78° C.→rt; (e) (1) N-iodosuccinimide,AgNO₃, (CH₃)₂CO; (2) Cy₂BH, Et₂O, AcOH; (f) (1) PhSH, BF₃.OEt₂, CH₂Cl₂,rt; (2) Ac₂O, pyridine, 4-DMAP, CH₂Cl₂, rt.

[0017]FIG. 4(B) presents synthesis of compound 1. (a) 11, 9-BBN, THF,rt; then PdCl₂(dppf)₂, Cs₂CO₃, Ph₃As, H₂O, DMF, 19, rt, 71%; (b) p-TsOH,dioxane/H₂O, 50° C.; (c) KHMDS, THF, −78° C., 51%; (d) (1) HF-pyridine,pyridine, THF, rt, 97%; (2) t-BuMe₂ SiOTf, 2,6-lutidine, CH₂Cl₂, −25°C., 93%; (3) Dess-Martin periodinane, CH₂Cl₂, 87%; (4) HF.pyridine, THF,rt, 99%; (e) dimethyldioxirane, CH₂Cl₂, 0.5 h, −50° C., 45% (≧20: 1).

[0018]FIG. 5 shows a scheme of the synthesis of the “left wing” ofepothilone A.

[0019]FIG. 6 provides a scheme of an olefin metathesis route toepothilone A and other analogues.

[0020]FIG. 7 illustrates a convergent strategy for a total synthesis ofepothilone A (1) and the glycal cyclopropane solvolysis strategy for theintroduction of geminal methyl groups.

[0021]FIG. 8 provides an enantioselective synthesis of compound 15B.

[0022]FIG. 9 shows the construction of epothilone model systems 20B,21B, and 22B by ring-closing olefin metathesis.

[0023]FIG. 10 illustrates a sedimentation test for natural, syntheticand desoxyepothilone A.

[0024]FIG. 11 illustrates a sedimentation test for natural, syntheticand desoxyepothilone A after cold treatment at 4° C.

[0025]FIG. 12 illustrates (A) structures of epothilones A (1) and B (2)and (B) of Taxol® (1A).

[0026]FIG. 13 shows a method of elaborating acyclic stereochemicalrelationships based on dihydropyrone matrices.

[0027]FIG. 14 shows the preparation of intermediate 4A.

[0028]FIG. 15 shows an alternative enantioselective synthesis ofcompound 17A.

[0029]FIG. 16 provides a synthetic pathway to intermediate 13C. (a) 1.tributyl allyltin, (S)-(−)-BINOL, Ti(Oi-Pr)₄, CH₂Cl₂, −20° C., 60%, >95%e.e.; 2. Ac₂O, Et₃N, DMAP, CH₂Cl₂, 95%; (b) 1. OsO₄, NMO, acetone/H₂O,0° C.; 2. NaIO₄, THF/H₂O; (c) 12, THF, −20° C., Z isomer only, 25% from10; (d) Pd(dppf)₂, Cs₂CO₃, Ph₃As, H₂O, DMF, rt. 77%.

[0030]FIG. 17 provides a synthetic pathway to intermediate epothilone B(2). (a) p-TsOH, dioxane/H₂O, 55° C., 71%; (b) KHMDS, THF, −78° C., α/β:1.5:1; (c) Dess-Martin periodinane, CH₂Cl₂; (d) NaBH₄, MeOH, 67% for twosteps; (e) 1. HF.pyridine, pyridine, THF, rt, 93%; 2. TBSOTf,2,6-lutidine, CH₂Cl₂, −30° C., 89%; 3. Dess-Martin periodinane, CH₂Cl₂,67%; (f) HF.pyridine, THF, rt, 80%; (g) dimethyldioxirane, CH₂Cl₂, −50°C., 70%.

[0031]FIG. 18 provides a synthetic pathway to a protected intermediatefor 8-desmethyl deoxyepothilone A.

[0032]FIG. 19 provides a synthetic pathway to 8-desmethyldeoxyepothilone A, and structures of trans-8-desmethyl-desoxyepothioloneA and a trans-iodoolefin intermediate thereto.

[0033]FIG. 20 shows (top) structures of epothilones A and B and8-desmethylepothilone and (bottom) a synthetic pathway to intermediateTBS ester 10 used in the preparation of desmethylepothilone A. (a)(Z)-Crotyl-B[(−)-Ipc]₂, −78° C., Et₂O, then 3N NaOH, 30% H₂O₂; (b)TBSOTf, 2,6-lutidine, CH₂Cl₂ (74% for two steps, 87% ee); (c) 03,CH₂Cl₂/MeOH, −78° C., then DMS, (82%); (d) t-butyl isobutyrylacetate,NaH, BuLi, 0° C., then 6 (60%, 10:1); (e) Me₄NBH(OAc)₃, −10° C. (50%,10:1 α/β) or NaBH₄, MeOH, THF, 0° C., (88%, 1:1 α/β; (f) TBSOTf,2,6-lutidine, −40° C., (88%); (g) Dess-Martin periodinane, (90%); (h)Pd(OH)₂, H₂, EtOH (96%); (I) DMSO, oxalyl chloride, CH₂Cl₂, −78° C.(78%); (j) Methyl triphenylphosphonium bromide, NaHMDS, THF, 0° C.(85%); (k)TBSOTf, 2,6-lutidine, CH₂Cl₂, rt (87%).

[0034]FIG. 21 shows a synthetic pathway to 8-desmethylepothilone A. (a)Pd(dppf)₂Cl₂, Ph₃As, Cs₂CO₃, H₂O, DMF, rt (62%); (b) K₂CO₃, MeOH, H₂O(78%); (c) DCC, 4-DMAP, 4-DMAP.HCl, CHCl₃ (78%); (d) HF.pyr, THF, rt(82%), (e) 3,3-dimethyl dioxirane, CH₂Cl₂, −35° C.(72%, 1.5:1).

[0035]FIG. 22 shows a synthetic pathway to prepare epothilone analogue27D.

[0036]FIG. 23 shows a synthetic pathway to prepare epothilone analogue24D.

[0037]FIG. 24 shows a synthetic pathway to prepare epothilone analogue19D.

[0038]FIG. 25 shows a synthetic pathway to prepare epothilone analogue20D.

[0039]FIG. 26 shows a synthetic pathway to prepare epothilone analogue22D.

[0040]FIG. 27 shows a synthetic pathway to prepare epothilone analogue12-hydroxy ethyl-epothilone.

[0041]FIG. 28 shows the activity of epothilone analogues in asedimentation test in comparison with DMSO, epothilone A and/or B.Structures 17-20, 22, and 24-27 are shown in FIGS. 29-37, respectively.Compounds were added to tubulin (1 mg/ml) to a concentration of 10 μM.The quantity of microtubules formed with epothilone A was defined as100%.

[0042]FIG. 29 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #17.

[0043]FIG. 30 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #18.

[0044]FIG. 31 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #19.

[0045]FIG. 32 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #20.

[0046]FIG. 33 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #22.

[0047]FIG. 34 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #24.

[0048]FIG. 35 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #25.

[0049]FIG. 36 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #26.

[0050]FIG. 37 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #27.

[0051]FIG. 38 provides a graphical representation of the effect offractional combinations of cytotoxic agents.

[0052]FIG. 39 shows epothilone A and epothilone analogues #1-7.Potencies against human leukemia CCRF-CEM (sensitive) and CCRF-CEM/VBLMDR (resistant) sublines are shown in round and square brackets,respectively.

[0053]FIG. 40 shows epothilone B and epothilone analogues #8-16.Potencies against human leukemia CCRF-CEM (sensitive) and CCRF-CEM/VBLMDR (resistant) sublines are shown in round and square brackets,respectively.

[0054]FIG. 41 shows epothilone analogues #17-25. Potencies against humanleukemia CCRF-CEM (sensitive) and CCRF-CEM/VBL MDR (resistant) sublinesare shown in round and square brackets, respectively.

[0055]FIG. 42(A) shows epothilone analogues #26-34. Potencies againsthuman leukemia CCRF-CEM (sensitive) and CCRF-CEM/VBL MDR (resistant)sublines are shown in round and square brackets, respectively. (B) showsepothilone analogues #35-46.Potencies against human leukemia CCRF-CEM(sensitive) and CCRF-CEM/VBL MDR (resistant) sublines are shown in roundand square brackets, respectively. (C) shows epothilone analogues#47-49.

[0056]FIG. 43(A) shows antitumor activity of desoxyepothilone B againstMDR MCF-7/Adr xenograft in comparison with Taxol®. Control (♦);desoxyepothilone B (▪; 35 mg/kg); Taxol® (▴; 6 mg/kg); adriamycin (×;1.8mg/kg); i.p. Q2Dx5; start on day 8. (B) shows antitumor activity ofepothilone B against MDR MCF-7/Adr xenograft in comparison with Taxol®.Control (♦); epothilone B (▪; 25 mg/kg; non-toxic dose); Taxol® (▴; 6mg/kg; half LD₅1); adriamycin (×;1.8 mg/kg); i.p. Q2Dx5; start on day 8.

[0057]FIG. 44(A) shows toxicity of desoxyepothilone B in B6D2F, micebearing B16 melanoma. Body weight was determined at 0, 2, 4, 6, 8, 10and 12 days. Control (A); desoxyepothilone B (∘; 10 mg/kg QDx8; 0 of 8died); desoxyepothilone B (; 20 mg/kg QDx6; 0 of 8 died). Injectionswere started on day 1. (B) shows toxicity of epothilone B in B6D2F, micebearing B16 melanoma. Body weight was determined at 0, 2, 4, 6, 8, 10and 12 days. Control (▴); epothilone B (∘; 0.4 mg/kg QDx6; 1 of 8 diedof toxicity); epothilone B (; 0.8 mg/kg QDx5; 5 of 8 died). Injectionswere started on day 1.

[0058]FIG. 45(A) shows comparative therapeutic effect ofdesoxyepothilone B and Taxol® on nude mice bearing MX-1 xenoplant.Tumor, s.c.; drug administered i.p., Q2Dx5, start on day 7. control (♦);Taxol® (□; 5 mg/kg, one half of LD₅₀); desoxyepothilone B (Δ; 25 mg/kg;nontoxic dose). (B) shows comparative therapeutic effect ofdesoxyepothilone B and Taxol® on nude mice bearing MX-1 xenoplant.Tumor, s.c.; drug administered i.p., Q2Dx5, start on day 7. control (♦);Taxol® (□; 5 mg/kg, one half of LD₅, given on days 7, 9, 11, 13, 15;then 6 mg/kg, given on days 17, 19, 23, 24, 25); desoxyepothilone B(n=3; Δ, ×, *; 25 mg/kg, nontoxic dose, given to three mice on days 7,9, 11, 13, 15; then 35 mg/kg, given on days 17, 19, 23, 24, 25).

[0059]FIG. 46 shows the effect of treatment with desoxyepothilone B (35mg/kg), Taxol® (5 mg/kg) and adriamycin (2 mg/kg) of nude mice bearinghuman MX-1 xenograft on tumor size between 8 and 18 days afterimplantation. Desoxyepothilone B (□), Taxol® (Δ), adriamycin (×),control (♦); i.p. treatments were given on day 8, 10, 12, 14 and 16.

[0060]FIG. 47 shows the relative toxicity of epothilone B (□; 0.6 mg/kgQDx4; i.p.) and desoxyepothilone B (Δ; 25 mg/kg QDx4; i.p.) versuscontrol (♦) in normal nude mice. Body weight of mice was determineddaily after injection. For epothilone B, 8 of 8 mice died of toxicity ondays 5, 6, 6, 7, 7, 7, 7, and 7; for desoxyepothilone B, all six micesurvived.

[0061]FIG. 48 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #43.

[0062]FIG. 49 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #45.

[0063]FIG. 50 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #46.

[0064]FIG. 51 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #47.

[0065]FIG. 52 shows a high resolution ¹H NMR spectrum of epothiloneanalogue #48.

[0066]FIG. 53(A) shows an approach to the preparation ofdesoxyepothilones. (B) represents a key step involving dianion additionto an aldehyde reactant.

[0067]FIG. 54(A) illustrates the acylation of t-butyl4-methylpentan-3-on-1-ate to provide t-butyl4,4-dimethylheptan-3,5-dion-1-ate. (B) exemplifies a dianion addition to2-methylpent-4-enal, diacylation and selective saponification to form akey intermediate 11-carbon diketoester.

[0068]FIG. 55 shows the preparation of a key intermediate hydroxyacid inthe preparation of desoxyepothilones. The final synthetic steps leadingto various desoxyepothilones from the hydroxyacid are found in, e.g.,FIGS. 21-26. R is selected from the group consisting of H, Me, Et, Pr,Hx, CH₂OH and (CH₂)₃OH. Ar is selected from the group consisting ofphenyl, tolyl, xylyl, thiazolyl, 2-methylthiazolyl, pyrryl and pyridyl,and is either unsubstituted or substituted with an C₁₋₆ alkyl, phenyl orbenzyl group. Conditions for the Noyori reduction are disclosed in Taberet al., Tetrahedron Lett., 1991, 32, 4227, and Noyori et al., J. Amer.Chem. Soc., 1987, 109, 5856, the contents of which are incorporatedherein by reference. Conditions for the DDQ deprotection are disclosedin Horita et al., Tetrahedron, 1986, 42, 3021, the contents of which isincorporated herein by reference.

[0069]FIG. 56 illustrates an application of the Noyori reduction of asubstrate with C-15 hydroxyl useful in the preparation of epothiloneanalogues.

[0070]FIG. 57 exemplifies a dianion addition to a coupled aldehydeintermediate useful in the preparation of epothilone analogues.

[0071]FIG. 58 provides an application of the Noyori reduction of acoupled substrate useful in the preparation of epothilone analogues.

[0072]FIG. 59 shows the preparation of desoxyepothilone analogues bydeoxygenation of the epoxide using Zn/Cu couple, exemplified by theconversion of desoxyepothilone B from epothilone B. In a sampleprocedure, Zn/Cu couple is added to a solution of epothilone B (6 mg,0.012 mmol) in i-PrOH (0.3 mL) and water (3 drops). The suspension washeated to 90° C. for 13 hours, cooled to room temperature, filteredthrough a pad of Celite™ and concentrated. Flash chromatography afforded1.5 mg of epothilone B (75% conversion) and 3.2 mg of desoxyepothilone B(73% yield, as a mixture of cis and trans isomers in a 0.7:1 ratio).

[0073]FIG. 60 illustrates the therapeutic effect of dEpoB, Taxol® andadriamycin in nude mice bearing the human mammary carcinoma MX-1xenograft. MX-1 tissue preparation 100 μl/mouse was implanted s.c. onday 0. Every other day i.p. treatments were given on day 8, 10, 12, 14and 16 with dEpoB 35 mg/kg (▪), Taxol® 5 mg/kg (▴), adriamycin (2 mg/kg(×), and vehicle (DMSO, 30 μl) treated control (♦). For Taxol®, 2/10mice died of toxicity on day 18. For adriamycin, 1/10 mice died oftoxicity on day 22. For dEpoB, 10/10 mice survived and were subjected tothe second cycle of treatment at 40 mg/kg on day 18, 20, 22, 24 and 26.This led to 3/10 mice tumor-free up to day 80, whereas 7/10 mice werewith markedly suppressed tumors and were sacrificed on day 50.

[0074]FIG. 61(A) shows a procedure for preparing intermediate 49.^(a)NaH, THF, 25° C., then 0° C., then propionyl chloride, −50° C., 71%;^(b)NaH, 0° C. then TESOTf, −50° C., 78%; LDA, THF, −33° C., 5 min.

[0075]FIG. 61(B) shows a procedure for preparing desoxyepothilone B._(a)TrocCl, pyridine, CH₂Cl₂, 0° C.→25° C.; then 0.5N HCl in MeOH, OC,87%; ^(b)9-BBN, THF, 50 then 51, (Pd(dppf)₂)Cl₂, Ph₃As, Cs₂CO₃, H₂O,DMF; ^(c)0.4N HCl in MeOH (50% for two steps); ^(d)(R)-(BINAP)RuCl₂, H₂(1200 psi), MeOH, HCl, 25° C., 7 h, 88%, >95:5; ^(e)TESOTf,2,6-lutidine, CH₂Cl₂, −78→25° C., then HCl/MeOH, 77%;^(f)2,4,6-trichlorobenzoyl chloride, TEA, 4-DMAP, toluene, 78%;^(g)SmI₂, cat. NiI₂, THF, −78° C., 95%; ^(h)HF.pyridine, THF, 98%;^(i)2,2-dimethyldioxirane, CH₂Cl₂, −50° C., 98%, >20:1.

[0076]FIG. 62 shows the therapeutic effect of dEpoB and Taxol® in nudemice bearing MX-1 with Q2Dx5 i.v. 6 h infusion, using dEpoB 30 mg/kg(×), Taxol® 15 mg/kg (▪), Taxol® 24 mg/kg (Δ) and control ().

[0077]FIG. 63 shows the therapeutic effect of dEpoB and Taxol® in nudemice bearing MCF-7/Adr with Q2Dx5 i.v. 6 h infusion, using dEpoB 30mg/kg (×), Taxol® 15 mg/kg (▪), Taxol® 24 mg/kg (Δ) and control ().

[0078]FIG. 64 shows the therapeutic effect of dEpoB and Taxol® in nudemice bearing CCRF/Taxol® with Q2Dx5 i.v. 6 h infusion, using dEpoB 30mg/kg (×), Taxol® 20 mg/kg (▪), Taxol® 24 mg/kg (Δ) and control ().Treatment on days 6, 8, 10,12 and 14.

[0079]FIG. 65 shows a procedure for preparing desoxyepothilone B (14E).

[0080]FIG. 66 shows a procedure for preparing intermediate 8E.

[0081]FIG. 67 shows the therapeutic effect of dEpoB and Taxol® in nudemice bearing CCRF/CEM tumor with Q2Dx4 i.v. 6 h infusion, using dEpoB 30mg/kg (□), Taxol® 20 mg/kg (Δ), and control (⋄). Treatment on days 21,23, 25, and 27.

[0082]FIG. 68 shows the therapeutic effect of dEpoB and Taxol® in nudemice bearing CCRF/Taxol® with Q2Dx5 i.v. 6 h infusion, using using dEpoB30 mg/kg (□), Taxol® 20 mg/kg Δ) and control (⋄). Treatment on days 19,21, 23, 25, 27, 39, 41, 43, 45 and 47.

[0083]FIG. 69 shows the therapeutic effect of dEpoB and Taxol® in nudemice bearing SK-OV-3 Tumor with Q2Dx6 i.v. 6 h infusion, using dEpoB 30mg/kg (Δ), Taxol® 15 mg/kg (□), and control (⋄). Treatment on days 10,12, 14, 16, 18 and 20.

[0084]FIG. 70 shows changes in body weight following treatment withdesoxyepothilone B and Taxol® in nude mice bearing SK-OV-3 Tumor by i.v.infusion, using dEpoB 30 mg/kg (□), Taxol® 15 mg/kg (Δ) and control (⋄).

[0085]FIG. 71 shows the therapeutic effect of dEpoB and Taxol® in nudemice bearing PC-3 Human Prostate Carcinoma with Q2Dx3 i.v. 6 or 18 hinfusion, using dEpoB 30 mg/kg, 18 h (×), dEpoB 40 mg/kg, 6 h (*), dEpoB50 mg/kg, 6 h (∘) Taxol® 15 mg/kg, 6 h (□), Taxol® 24 mg/kg, 6 h (Δ) andcontrol (♦). Treatment on days 5, 7 and 9.

[0086]FIG. 72 shows the therapeutic effect of dEpoB and Taxol® in nudemice bearing CCRF-CEM/VBL with Q2Dx5 i.v. 6 h infusion, using usingdEpoB 30 mg/kg (□), Taxol® 20 mg/kg (Δ) and control (⋄). Treatment ondays 19, 21, 23, 25, 27, 39, 41, 43, 45, 47 and 53.

[0087]FIG. 73 shows the therapeutic effect of dEpoB and Taxol® in nudemice bearing HT-29 Colon Adenocarcinoma with Q2Dx6 i.v. 6 h infusion,using dEpoB 30 mg/kg (Δ), Taxol® 15 mg/kg (□) and control (⋄).

[0088]FIG. 74 shows the therapeutic effect of dEpoB and Taxol® in nudemice bearing A549 Human Lung Carcinoma with Q2Dx3 i.v. 6 or 18 hinfusion, using dEpoB 30 mg/kg 18 h (×), dEpoB 40 mg/kg, 6 h (*), dEpoB50 mg/kg, 6 h (∘), Taxol® 15 mg/kg, 6 h (□), Taxol® 24 mg/kg, 6 h (Δ)and control (♦). Treatment on days 7, 9 and 11.

[0089]FIG. 75 shows the Epi stability in plasma of dEpoB human (Δ) andmice ().

DETAILED DESCRIPTION OF THE INVENTION

[0090] As used herein, the term “linear or branched chain alkyl”encompasses, but is not limited to, methyl, ethyl, propyl, isopropyl,t-butyl, sec-butyl, cyclopentyl or cyclohexyl. The alkyl group maycontain one carbon atom or as many as fourteen carbon atoms, butpreferably contains one carbon atom or as many as nine carbon atoms, andmay be substituted by various groups, which include, but are not limitedto, acyl, aryl, alkoxy, aryloxy, carboxy, hydroxy, carboxamido and/orN-acylamino moieties.

[0091] As used herein, the terms “alkoxycarbonyl”, “acyl” and “alkoxy”encompass, but are not limited to, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, n-butoxycarbonyl, benzyloxycarbonyl,hydroxypropylcarbonyl, aminoethoxycarbonyl, sec-butoxycarbonyl andcyclopentyloxycarbonyl. Examples of acyl groups include, but are notlimited to, formyl, acetyl, propionyl, butyryl and penanoyl. Examples ofalkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy,n-butoxy, sec-butoxy and cyclopentyloxy.

[0092] As used herein, an “aryl” encompasses, but is not limited to, aphenyl, pyridyl, pyrryl, indolyl, naphthyl, thiophenyl or furyl group,each of which may be substituted by various groups, which include, butare not limited, acyl, aryl alkoxy, aryloxy, carboxy, hydroxy,carboxamido or N-acylamino moieties. Examples of aryloxy groups include,but are not limited to, a phenoxy, 2-methylphenoxy, 3-methylphenoxy and2-naphthoxy. Examples of acyloxy groups include, but are not limited to,acetoxy, propanoyloxy, butyryloxy, pentanoyloxy and hexanoyloxy.

[0093] The subject invention provides chemotherapeutic analogues ofepothilone A and B, including a compound having the structure:

[0094] wherein R, R₀, and R′ are independently H, linear or branchedchain alkyl, optionally substituted by hydroxy, alkoxy, fluorine, NR₁R₂,N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ are independently H,phenyl, benzyl, linear or branched chain alkyl; wherein R″ is —CHY═CHX,or H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; and whereinX is H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein Zis O, N(OR₃) or N—NR₄R₅, wherein R₃, R₄ and R₅ are independently H or alinear or branched alkyl; and wherein n is 0, 1, 2, or 3. In oneembodiment, the invention provides the compound having the structure:

[0095] wherein R is H, methyl, ethyl, n-propyl, n-butyl, n-hexyl, CH₂OH,or (CH₂)₃OH.

[0096] The invention also provides a compound having the structure:

[0097] wherein R, R₀, and R′ are independently H, linear or branchedchain alkyl, optionally substituted by hydroxy, alkoxy, fluorine, NR₁R₂,N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ are independently H,phenyl, benzyl, linear or branched chain alkyl; wherein R″ is —CHY═CHX,or H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; and whereinX is H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein Zis O, N(OR₃) or N—NR₄R₅, wherein R₃, R₄ and R₅ are independently H or alinear or branched chain alkyl; and wherein n is 0, 1, 2, or 3. In acertain embodiment, the invention provides a compound having thestructure:

[0098] wherein R is H, methyl, ethyl, n-propyl, n-butyl, n-hexyl orCH₂OH.

[0099] In addition, the invention provides a compound having thestructure:

[0100] wherein R, R₀, and R′ are independently H, linear or branchedchain alkyl, optionally substituted by hydroxy, alkoxy, fluorine, NR₁R₂,N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ are independently H,phenyl, benzyl, linear or branched chain alkyl; wherein R″ is —CHY═CHX,or H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; and whereinX is H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein Zis O, N(OR₃) or N—NR₄R₅, wherein R₃, R₄ and R₅ are independently H or alinear or branched chain alkyl; and wherein n is 0, 1, 2, or 3. Inparticular, the invention provides a compound having the structure:

[0101] wherein R is H, methyl, ethyl, n-propyl, n-butyl, CH₂OH or(CH₂)₃OH.

[0102] The invention further provides a compound having the structure:

[0103] wherein R, R₀, and R′ are independently H, linear or branchedchain alkyl, optionally substituted by hydroxy, alkoxy, fluorine, NR₁R₂,N-hydroximino or N-alkoxyimino, wherein R₁ and R₂ are independently H,phenyl, benzyl, linear or branched chain alkyl; wherein R″ is —CHY═CHX,or H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; and whereinX is H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein Zis O, N(OR₃) or N—NR₄R₅, wherein R3, R₄ and R₅ are independently H or alinear or branched chain alkyl; and wherein n is 0, 1, 2 or 3.

[0104] The invention also provides a compound having the structure:

[0105] The subject invention also provides various intermediates usefulfor the preparation of the chemotherapeutic compounds epithilone A andB, as well as analogues thereof. Accordingly, the invention provides akey intermediate to epothilone A and its analogues having the structure:

[0106] wherein R is hydrogen, a linear or branched acyl, substituted orunsubstituted aroyl or benzoyl; wherein R′ is hydrogen, methyl, ethyl,n-propyl, n-hexyl,

[0107] CH₂OTBS or (CH₂)₃—OTBDPS; and X is a halide. In one embodiment,the subject invention provides a compound of the above structure whereinR is acetyl and X is iodo.

[0108] The subject invention also provides an intermediate having thestructure:

[0109] wherein R′ and R″ are independently hydrogen, a linear orbranched alkyl, substituted or unsubstituted aryl or benzyl, trialkylsilyl, dial kylarylsilyl, alkyldiarylsilyl, a linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl; wherein X isoxygen, (OR)₂, (SR)₂, —(O—(CH₂)_(n)—O)—, —(O—(CH₂)_(n)—S)— or—(S—(CH₂)_(n)—S)—; and wherein n is 2, 3 or 4.

[0110] wherein R is H or methyl.

[0111] Another analogue provided by the invention has the structure:

[0112] wherein R is H, methyl, ethyl, n-propyl, n-butyl, n-hexyl, CH₂OH,or (CH₂)₃OH.

[0113] Additionally, the subject invention provides an analogue havingthe structure:

[0114] wherein R is H or methyl. The scope of the present inventionincludes compounds wherein the C₃ carbon therein possesses either an Ror S absolute configuration, as well as mixtures thereof.

[0115] The subject invention further provides an analogue of epothiloneA having the structure:

[0116] The subject invention also provides synthetic routes to preparethe intermediates for preparing epothilones. Accordingly, the inventionprovides a method of preparing a Z-iodoalkene ester having thestructure:

[0117] wherein R is hydrogen, a linear or branched alkyl, alkoxyalkyl,substituted or unsubstituted aryloxyalkyl, linear or branched acyl,substituted or unsubstituted aroyl or benzoyl, which comprises (a)coupling a compound having the structure:

[0118] with a methyl ketone having the structure

[0119] wherein R′ and R″ are independently a linear or branched alkyl,alkoxyalkyl, substituted or unsubstituted aryl or benzyl, under suitableconditions to form a compound having the structure:

[0120] (b) treating the compound formed in step (a) under suitableconditions to form a Z-iodoalkene having the structure:

[0121] and (c) deprotecting and acylating the Z-iodoalkene formed instep (b) under suitable conditions to form the Z-iodoalkene ester. Thecoupling in step (a) may be effected using a strong base such as n-BuLiin an inert polar solvent such as tetrahydrofuran (THF) at lowtemperatures, typically below −50° C., and preferably at −78° C. Thetreatment in step (b) may comprise sequential reaction withN-iodosuccinimide in the presence of Ag(l), such as silver nitrate, in apolar organic solvent such as acetone, followed by reduction conditions,typically using a hydroborating reagent, preferably using Cy₂BH.Deprotecting step (c) involves contact with a thiol such as thiophenolin the presence of a Lewis acid catalyst, such as borontrifluoride-etherate in an inert organic solvent such asdichloromethane, followed by acylation with an acyl halide, such asacetyl chloride, or an acyl anhydride, such as acetic anhydride in thepresence of a mild base such as pyridine and/or 4-dimethyaminopyridine(DMAP) in an inert organic solvent such as dichloromethane.

[0122] The subject invention also provides a method of preparing aZ-haloalkene ester having the structure:

[0123] wherein R is hydrogen, a linear or branched alkyl, alkoxyalkyl,substituted or unsubstituted aryloxyalkyl, linear or branched acyl,substituted or unsubstituted aroyl or benzoyl; and wherein X is ahalogen, which comprises (a) oxidatively cleaving a compound having thestructure:

[0124] under suitable conditions to form an aldehyde intermediate; and(b) condensing the aldehyde intermediate with a halomethylene transferagent under suitable conditions to form the Z-haloalkene ester. In oneembodiment of the method, X is iodine. In another embodiment, the methodis practiced wherein the halomethylene transfer agent is Ph₃P═CHI or(Ph₃P⁻CH₂I)I⁻. Disubstituted olefins may be prepared using thehaloalkylidene transfer agent Ph₃P═CR′I, wherein R′ is hydrogen, methyl,ethyl, n-prop-yl, n-hexyl,

[0125] CO₂Et or (CH₂)₃OTBDPS. The oxidative step (a) can beperformedusing a mild oxidant such as osmium tetraoxide at temperatures of about0° C., followed by treatment with sodium periodate, or with leadtetraacetate/sodium carbonate, to complete the cleavage of the terminalolefin, and provide a terminal aldehyde. Condensing step (b) occurseffectively with a variety of halomethylenating reagents, such as Wittigreagents.

[0126] The subject invention further provides a method of preparing anoptically pure compound having the structure:

[0127] wherein R is hydrogen, a linear or branched alkyl, alkoxyalkyl,substituted or unsubstituted aryloxyalkyl, linear or branched acyl,substituted or unsubstituted aroyl or benzoyl, which comprises: (a)condensing an allylic organometallic reagent with an unsaturatedaldehyde having the structure:

[0128] under suitable conditions to form an alcohol, and, optionallyconcurrently therewith, optically resolving the alcohol to form anoptically pure alcohol having the structure:

[0129] (b) alkylating or acylating the optically pure alcohol formed instep (a) under suitable conditions to form the optically pure compound.In one embodiment of the method, the allylic organometallic reagent isan allyl(trialkyl)stannane. In another embodiment, the condensing stepis effected using a reagent comprising a titanium tetraalkoxide and anoptically active catalyst. In step (a) the 1,2-addition to theunsaturated aldehyde may be performed using a variety of allylicorganometallic reagents, typically with an allyltrialkylstannane, andpreferably with allyltri-n-butylstannane, in the presence of chiralcatalyst and molecular sieves in an inert organic solvent such asdichloromethane. Preferably, the method may be practiced using titaniumtetraalkoxides, such as titanium tetra-n-propoxide, and S-(−)BINOL asthe optically active catalyst. Alkylating or acylating step (b) iseffected using any typical alkylating agent, such as alkylhalide oralkyl tosylate, alkyl triflate or alkyl mesylate, any typical acylatingagent, such as acetyl chloride, acetic anhydride, benzoyl chloride orbenzoyl anhydride, in the presence of a mild base catalyst in an inertorganic solvent, such as dichloromethane.

[0130] The subject invention also provides a method of preparing anopen-chain aldehyde having the structure:

[0131] wherein R′ and R″ are independently hydrogen, a linear orbranched alkyl, substituted or unsubstituted aryl or benzyl,trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, a linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl, which comprises:(a) cross-coupling a haloolefin having the structure:

[0132] wherein R is a linear or branched alkyl, alkoxyalkyl, substitutedor unsubstituted aryloxyalkyl, trialkylsilyl, aryldialkylsilyl,diarylalkylsilyl, triarylsilyl, linear or branched acyl, substituted orunsubstituted aroyl or benzoyl, and X is a halogen, with a terminalolefin having the estructure:

[0133] wherein (OR′″)₂ is (OR₀)₂, (SR₀)₂, —(O—(CH₂)_(n)—O)—,—(O—(CH₂)_(n)—S)— or —(S—(CH₂)_(n)—S)— where R₀ is a linear or branchedalkyl, substituted or unsubstituted aryl or benzyl; and wherein n is 2,3 or 4, under suitable conditions to form a cross-coupled compoundhaving the structure:

[0134] wherein Y is CH(OR*)₂ where R* is a linear or branched alkyl,alkoxyalkyl, substituted or unsubstituted aryloxyalkyl; and (b)deprotecting the cross-coupled compound formed in step (a) undersuitable conditions to form the open-chain compound. Cross-coupling step(a) is effected using reagents known in the art which are suited to thepurpose. For example, the process may be carried out by hydroboratingthe pre-acyl component with 9-BBN. The resulting mixed borane may thenbe cross-coupled with an organometallic catalyst such as PdCl₂(dppf)₂,or any known equivalent thereof, in the presence of such ancillaryreagents as cesium carbonate and triphenylarsine. Deprotecting step (b)can be carried out with a mild acid catalyst such as p-tosic acid, andtypically in a mixed aqueous organic solvent system, such asdioxane-water. The open-chain compound can be cyclized using any of avariety of non-nucleophilic bases, such as potassiumhexamethyldisilazide or lithium diethyamide.

[0135] The subject invention also provides a method of preparing anepothilone having the structure:

[0136] which comprises: (a) deprotecting a cyclized compound having thestructure:

[0137] wherein R′ and R″ are independently hydrogen, a linear orbranched alkyl, substituted or unsubstituted aryl or benzyl,trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, a linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl, under suitableconditions to form a deprotected cyclized compound and oxidizing thedeprotected cyclized compound under suitable conditions to form adesoxyepothilone having the structure:

[0138] and (b) epoxidizing the desoxyepothilone formed in step (a) undersuitable conditions to form the epothilone. Deprotecting step (a) iseffected using a sequence of treatments comprising a catalyst such asHF-pyridine, followed by t-butyldimethylsilyl triflate in the presenceof a base such as lutidine. Dess-Martin oxidation and furtherdeprotection with a catalyst such as HF-pyridine provides thedesoxyepothilone. The latter compound can then be epoxidized in step (b)using any of a variety of epoxidizing agents, such acetic peracid,hydrogen peroxide, perbenzoic acid, m-chloroperbenzoic acid, butpreferably with dimethyldioxirane, in an inert organic solvent such asdichloromethane.

[0139] The subject invention further provides a method of preparing anepothilone precursor having the structure:

[0140] wherein R₁ is hydrogen or methyl; wherein X is O, or a hydrogenand OR″, each singly bonded to carbon; and wherein R₀, R′ and R″ areindependently hydrogen, a linear or branched alkyl, substituted orunsubstituted aryl or benzyl, trialkylsilyl, dialkylarylsilyl,alkyidiaryisilyl, a linear or branched acyl, substituted orunsubstituted aroyl or benzoyl, which comprises (a) coupling a compoundhaving the structure:

[0141] wherein R is an acetyl, with an aldehyde having the structure:

[0142] wherein Y is oxygen, under suitable conditions to form an aldolintermediate and optionally protecting the aldol intermediate undersuitable conditions to form an acyclic epthilone precursor having thestructure:

[0143] (b) subjecting the acylic epothilone precursor to conditionsleading to intramolecular olefin metathesis to form the epothiloneprecursor. In one embodiment of the method, the conditions leading tointramolecular olefin metathesis require the presence of anorganometallic catalyst. In a certain specific embodiment of the method,the catalyst contains Ru or Mo. The coupling step (a) may be effectedusing a nonnucleophilic base such as lithium diethylamide or lithiumdiisopropylamide at subambient temperatures, but preferably at about−78° C. The olefin metathesis in step (b) may be carried out using anycatalyst known in the art suited for the purpose, though preferablyusing one of Grubbs's catalysts.

[0144] In addition, the present invention provides a compound useful asan intermediate for preparing epothilones having the structure:

[0145] wherein R′ and R″ are independently hydrogen, a linear orbranched alkyl, substituted or unsubstituted aryl or benzyl,trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, a linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl; wherein X isoxygen, (OR*)₂, (SR*)₂, —(O—(CH₂)_(n)—O)—, —(O—(CH₂)_(n)—S)— or—(S—(CH₂)_(n)—S)—; wherein R* is a linear or branched alkyl, substitutedor unsubstituted aryl or benzyl; wherein R₂B is a linear, branched orcyclic boranyl moiety; and wherein n is 2, 3 or 4. In certainembodiments, the invention provides the compound wherein R′ is TBS, R″is TPS and X is (OMe)₂. A preferred example of R₂B is derived from9-BBN.

[0146] The invention also provides the compound having the structure:

[0147] wherein R′ and R″ are independently hydrogen, a linear orbranched alkyl, substituted or unsubstituted aryl or benzyl,trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, a linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl; wherein X isoxygen, (OR)₂, (SR)₂, —(O—(CH₂)_(n)—O)—, —(O—(CH₂)_(n)—S)— or—(S—(CH₂)_(n)—S)—; and wherein n is 2, 3 or 4. In certain embodiments,the invention provides the compound wherein R′ is TBS, R″ is TPS and Xis (OMe)₂.

[0148] The invention further provides a desmethylepothilone analogouehaving the structure:

[0149] wherein R is H or methyl.

[0150] The invention provides a compound having the structure:

[0151] wherein R is H or methyl.

[0152] The invention also provides a trans-desmethyldeoxyepothiloneanalogue having the structure:

[0153] wherein R is H or methyl.

[0154] The invention also provides a trans-epothilone having thestructure:

[0155] wherein R is H or methyl.

[0156] The invention also provides a compound having the structure:

[0157] wherein R is hydrogen, a linear or branched alkyl, alkoxyalkyl,substituted or unsubstituted aryloxyalkyl, linear or branched acyl,substituted or unsubstituted aroyl or benzoyl; wherein R′ is hydrogen,methyl, ethyl, n-propyl, n-hexyl,

[0158] CO₂Et or (CH₂)₃OTBDPS. and X is a halogen. In certainembodiments, the invention provides the compound wherein R is acetyl andX is iodine.

[0159] The invention additionally provides a method of preparing anopen-chain aldehyde having the structure:

[0160] wherein R is a linear or branched alkyl, alkoxyalkyl, substitutedor unsubstituted aryloxyalkyl, trialkylsilyl, aryidialkylsilyl,diarylalkylsilyl, triarylsilyl, linear or branched acyl, substituted orunsubstituted aroyl or benzoyl; and wherein R′ and R are independentlyhydrogen, a linear or branched alkyl, substituted or unsubstituted arylor benzyl, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, a linearor branched acyl, substituted or unsubstituted aroyl or benzoyl, whichcomprises:

[0161] (a) cross-coupling a haloolefin having the structure:

[0162] wherein X is a halogen, with a terminal borane having thestructure:

[0163] wherein R*₂B is a linear, branched or cyclic alkyl or substitutedor unsubstituted aryl or benzyl boranyl moiety; and wherein Y is (OR₀)₂,(SR₀)₂, —(O—(CH₂)_(n)—O)—, —(O—(CH₂)_(n)—S)— or —(S—(CH₂)_(n)—S)— whereR₀ is a linear or branched alkyl, substituted or unsubstituted aryl orbenzyl; and wherein n is 2, 3 or 4, under suitable conditions to formacross-coupled compound having the structure:

[0164] and

[0165] (b) deprotecting the cross-coupled compound formed in step (a)under suitable conditions to form the open-chain aldehyde. In certainembodiments, the invention provides the method wherein R is acetyl; R′is TBS; R″ is TPS; R*₂B is derived from 9-BBN; and Y is (OMe)₂.Cross-coupling step (a) is effected using reagents known in the artwhich are suited to the purpose. For example, the mixed borane may becross-coupled with an organometallic catalyst such as PdCl₂(dppf)₂, orany known equivalent thereof, in the presence of such reagents as cesiumcarbonate and triphenylarsine. Deprotecting step (b) can be carried outusing a mild acid catalyst such as p-tosic acid, typically in a mixedaqueous organic solvent system, such as dioxane-water.

[0166] The invention also provides a method of preparing a protectedepothilone having the structure:

[0167] wherein R′ and R″ are independently hydrogen, a linear orbranched alkyl, substituted or unsubstituted aryl or benzyl,trialkylsilyl, dialkyl-arylsilyl, alkyldiarylsilyl, a linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl, which comprises:

[0168] (a) monoprotecting a cyclic diol having the structure:

[0169] under suitable conditions to form a cyclic alcohol having thestructure:

[0170] and

[0171] (b) oxidizing the cyclic alcohol formed in step (a) undersuitable conditions to form the protected epothilone. In certainembodiments, the invention provides the method wherein R′ and R″ areTBS. The monoprotecting step (a) may be effected using any of a varietyof suitable reagents, including TBSOTf in the presence of a base in aninert organic solvent. The base may be a non-nucleophilic base such as2,6-lutidine, and the solvent may be dichloromethane. The reaction isconducted at subambient temperatures, preferably in the range of −30° C.The oxidizing step (b) utilizes a selective oxidant such as Dess-Martinperiodinane in an inert organic solvent such as dichloromethane. Theoxidation is carried out at ambient temperatures, preferably at 20-25°C.

[0172] The invention further provides a method of preparing anepothilone having the structure:

[0173] which comprises:

[0174] (a) deprotecting a protected cyclic ketone having the structure:

[0175] wherein R′ and R″ are independently hydrogen, a linear orbranched alkyl, substituted or unsubstituted aryl or benzyl, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, a linear or branched acyl,substituted or unsubstituted aroyl or benzoyl, under suitable conditionsto form a desoxyepothilone having the structure:

[0176] and (b) epoxidizing the desoxyepothilone formed in step (a) undersuitable conditions to form the epothilone. In certain embodiments, theinvention provides the method wherein R′ and R″ are TBS. Deprotectingstep (a) is carried out by means of a treatment comprising a reagentsuch as HF.pyridine. The deprotected compound can be epoxidized in step(b) using an epoxidizing agent such acetic peracid, hydrogen peroxide,perbenzoic acid, m-chloroperbenzoic acid, but preferably withdimethyldioxirane, in an inert organic solvent such as dichloromethane.

[0177] The invention also provides a method of preparing a cyclic diolhaving the structure:

[0178] wherein R′ is a hydrogen, a linear or branched alkyl, substitutedor unsubstituted aryl or benzyl, trialkylsilyl, dialkylarylsilyl,alkyldiarylsilyl, a linear or branched acyl, substituted orunsubstituted aroyl or benzoyl, which comprises:

[0179] (a) cyclizing an open-chain aldehyde having the structure:

[0180] wherein R is a linear or branched alkyl, alkoxyalkyl, substitutedor unsubstituted aryloxyalkyl, trialkylsilyl, aryldialkylsilyl,diarylalkylsilyl, triarylsilyl, linear or branched acyl, substituted orunsubstituted aroyl or benzoyl; and wherein R is a hydrogen, a linear orbranched alkyl, substituted or unsubstituted aryl or benzyl,trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, a linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl under suitableconditions to form an enantiomeric mixture of a protected cyclic alcoholhaving the structure:

[0181] said mixture comprising an α- and a β-alcohol component;

[0182] (b) optionally isolating and oxidizing the α-alcohol formed instep (a) under suitable conditions to form a ketone and thereafterreducing the ketone under suitable conditions to form an enantiomericmixture of the protected cyclic alcohol comprising substantially theβ-alcohol; and

[0183] (c) treating the protected cyclic alcohol formed in step (a) or(b) with a deprotecting agent under suitable conditions to form thecyclic diol. In certain embodiments, the invention provides the methodwherein R′ is TBS and R″ is TPS. Cyclizing step (a) is performed usingany of a variety of mild nonnucleophilic bases such as KHMDS in an inertsolvent such as THF. The reaction is carried out at subambienttemperatures, preferably between −9⁰° C. and −50° C., more preferably at−78° C. Isolation of the unnatural alpha-OH diastereomer is effected byany usual purification method, including any suitable type ofchromatography or by crystallization. Chromatographic techniques usefulfor the purpose include high pressure liquid chromatography,countercurrent chromatography or flash chromatography. Various columnmedia are suited, including, inter alia, silica or reverse phasesupport. The beta-OH derivative is then oxidized using a selectiveoxidant, such as Dess-Martin periodinane. The resulting ketone is thereduced using a selective reductant. Various hydridoborane and aluminumhydride reagents are effective. A preferred reducing agent is sodiumborohydride. Treating step (c ) may be effected using a variety ofdeprotecting agents, including HF-pyridine.

[0184] The present invention provides a method of preparing adesoxyepothilone having the structure:

[0185] wherein R, R₀, and R′ are independently H, linear or branchedchain alkyl, optionally substituted by hydroxy, alkoxy, carboxy,carboxaldehyde, linear or branched alkyl or cyclic acetal, fluorine,NR₁R₂, N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ areindependently H, phenyl, benzyl, linear or branched chain alkyl; whereinR″ is —CY═CX—, or H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein X is H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein Zis O, N(OR₃) or N—NR₄R₅, wherein R₃, R₄ and R₅ are independently H or alinear or branched alkyl; and wherein n is 0, 1, 2, or 3; whichcomprises treating an epothilone having a structure:

[0186] wherein R, R₀, R₁, R₂, R₃, R₄, R₅, R′, R″, X, Y, Z and n aredefined as for the desoxyepothilone, under suitable conditions so as todeoxygenate the epothilone, and thereby to provide the desoxyepothilone.In one embodiment, the method is effected wherein the desoxyepothilonehas the structure:

[0187] wherein R is H, methyl, ethyl, n-propyl, n-butyl, n-hexyl,

[0188] or (CH₂)₃—OH. In another embodiment, the method is effectedwherein the epothilone is deoxygenated using a zinc/copper couple.Preferably, the method is carried out wherein the epothilone isdeoxygenated in the presence of a polar solvent comprising isopropanoland water.

[0189] The present invention further provides a method of preparing adesoxyepothilone having the structure:

[0190] wherein R, R₀, and R′ are independently H, linear or branchedchain alkyl, optionally substituted by hydroxy, alkoxy, carboxy,carboxaldedyde, linear or branched alkyl or cyclic acetal, fluorine,NR1R₂, N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ areindependently H, phenyl, benzyl, linear or branched chain alkyl; whereinR″ is —CY═CHX, or H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 34uranyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein X is H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein Zis O, N(OR₃) or N—NR₄R₅, wherein R₃, R₄ and R₅ are independently H or alinear or branched chain alkyl; and wherein n is 0, 1, 2, or 3; whichcomprises treating an epothilone having a structure:

[0191] wherein R, R₀, R₁, R₂, R₃, R₄, R₅, R′, R″, X, Y, Z and n aredefined as for the desoxyepothilone, under suitable conditions so as todeoxygenate the epothilone, and thereby to provide the desoxyepothilone.In one embodiment, the method is performed wherein the desoxyepothilonehas the structure:

[0192] wherein R is H, methyl, ethyl, n-propyl, n-butyl, n-hexyl orhydroxypropyl. Preferably, the method is effected wherein the epothiloneis deoxygenated using a zinc/copper couple. Favorably, the epothilone isdeoxygenated in the presence of a polar solvent comprising isopropanoland water.

[0193] The present invention also provides a method of preparing adesoxyepothilone having the structure:

[0194] wherein R, R₀, and R′ are independently H, linear or branchedchain alkyl, optionally substituted by hydroxy, alkoxy, carboxy,carboxaldedyde, linear or branched alkyl or cyclic acetal, fluorine,NR₁R₂, N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ areindependently H, phenyl, benzyl, linear or branched chain alkyl; whereinR″ is —CY═CHX, or H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein X is H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein Y is H or linear or branched chain alkyl; and whereinZ is O, N(OR₃) or N—NR₄R₅ where R₃, R₄ and R₅ are independently H or alinear or branched alkyl; and wherein n is 0, 1, 2, or 3; whichcomprises treating a protected desoxyepothilone having the structure:

[0195] wherein R_(A) is a linear or branched alkyl, alkoxyalkyl,substituted or unsubstituted aryloxyalkyl, trialkylsilyl,aryldialkylsilyl, diarylalkylsilyl, triarylsilyl, linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl; and wherein R_(B)is hydrogen, t-butyloxycarbonyl, amyloxycarbonyl,(trialkylsilyl)alkyloxycarbonyl, (dialkylarylsilyl) alkyloxycarbonyl,benzyl, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarysilyl,a linear or branched acyl, substituted or unsubstituted aroyl orbenzoyl; under suitable conditions to form the desoxyepothilone. In acertain embodiment, the invention provides a method wherein n is 3 andR″ is 2-methyl-1,3-thiazolinyl. Preferably, the method is effectedwherein R_(A) is TES and R_(B) is Troc. In a certain other embodiment,the invention provides a method wherein the treating step comprisescontacting the protected desoxyepothilone (i) with SmX₂, where X is Cl,Br or I, in the presence of a polar nonaqueous solvent selected from thegroup consisting of tetrahydrofuran, p-dioxane, diethyl ether,acetonitrile and N,N-dimethylformamide, and optionally in the presenceof N,N-dimethyl-N′-propylurea or hexamethylphosphoramide and (ii) with asource of fluoride ion selected from the group consisting oftetra-n-methylammonium fluoride, tetra-n-butylammonium fluoride andHF.pyridine.

[0196] The present invention also provides a method of preparing aprotected desoxyepothilone having the structure:

[0197] wherein R, R₀, and R′ are independently H, linear or branchedchain alkyl, optionally substituted by hydroxy, alkoxy, carboxy,carboxaldehyde, linear or branched alkyl or cyclic acetal, fluorine,NR₀R₂, N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ areindependently H, phenyl, benzyl, linear or branched chain alkyl; whereinR″ is —CY═CHX, or H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein X is H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein nis 2 or 3; wherein R_(A) is a linear or branched alkyl, alkoxyalkyl,substituted or unsubstituted aryloxyalkyl, trialkylsilyl,aryidialkylsilyl, diarylalkylsilyl, triarylsilyl, linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl; and wherein R_(B)is hydrogen, t-butyloxycarbonyl, amyloxycarbonyl,(trialkylsilyl)alkyloxycarbonyl, (dialkylarylsilyl)alkyloxycarbonyl,benzyl, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarysilyl,a linear or branched acyl, substituted or unsubstituted aroyl orbenzoyl; which comprises cyclocondensing a hydroxy acid desoxyepothiloneprecursor having the structure:

[0198] wherein R, R₀, R_(A), R_(B), R′, R″ and n are defined as above;under suitable conditions to form the protected desoxyepothilone. Inparticular, the method is carried out wherein n is 3 and R″ is2-methyl-1,3-thiazolinyl. Preferably, R_(A) is a trialkylsilyl, and ismore preferably, TES. R_(B) is favorably trichloroethyloxycarbonyl(Troc). According to the method, the hydroxy acid desoxyepothi loneprecursor is cyclocondensed using a cyclocondensing reagent selectedfrom the group consisting of acetic anhydride, pentafluorophenol,2,4-dichlorobenzoyl chloride and, preferably, 2,4,6-trichlorobenzoylchloride. In addition, the hydroxyacid is favorably cyclocondensed using2,4,6-trichlorobenzoyl chloride in the presence of a tertiary amineselected from the group consisting of triethyl amine, tri-n-propylamine,diisopropylethylamine and diethyliso-propylamine, and optionally in thepresence of pyridine or N,N-dimethylaminopyridine.

[0199] The present invention further provides a method of preparing ahydroxy acid desoxyepothilone precursor having the structure:

[0200] wherein R, R₀, and R′ are independently H, linear or branchedchain alkyl, optionally substituted by hydroxy, alkoxy, carboxy,carboxaldehyde, linear or branched alkyl or cyclic acetal, fluorine,NR₁R₂, N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ areindependently H, phenyl, benzyl, linear or branched chain alkyl; whereinR″ is —CY═CHX, or H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein X is H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein nis 2 or 3; wherein R_(A) is a linear or branched alkyl, alkoxyalkyl,substituted or unsubstituted aryloxyalkyl, trialkylsilyl,aryldialkylsilyl, diarylalkylsilyl, triarylsilyl, linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl; wherein R_(B) ishydrogen, t-butyloxycarbonyl, amyloxycarbonyl, (trialkylsilyl)alkyloxycarbonyl, (dialkylarylsilyl)alkyloxycarbonyl, benzyl,trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, linearor branched acyl, substituted or unsubstituted aroyl or benzoyl; whichcomprises selectively etherifying and hydrolyzing a hydroxy esterdesoxyepothilone precursor having the structure:

[0201] wherein R, R₀, R_(B), R_(C), R′, R″ and n are defined as above;and wherein R_(C) is tertiary-alkyl; under suitable conditions to formthe hydroxy acid desoxyepothilone precursor. In one embodiment, theinvention is practiced wherein n is 3 and R″ is2-methyl-1,3-thiazolinyl. Preferably, R_(A) is TES and R_(B) is Troc. Inaccord with invention, the method is performed wherein the selectiveetherifying step comprises contacting the hydroxy ester desoxyepothiloneprecursor with a silylating reagent to form an ether intermediate, andthe hydrolyzing step comprises contacting the ether intermediate with aprotic acid or tetra-n-butylammonium fluoride. Favorably, the silylatingreagent is TESOTf in the presence of 2,6-lutidine. The protic acid istypically HCl in the presence of an alkyl alcohol, preferably, methylalcohol or ethyl alcohol.

[0202] The present invention also provides a method of preparing ahydroxy ester desoxyepothilone precursor having the structure:

[0203] wherein R, R₀, and R′ are independently H, linear or branchedchain alkyl, optionally substituted by hydroxy, alkoxy, carboxy,carboxaldehyde, linear or branched alkyl or cyclic acetal, fluorine,NR₁R₂, N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ areindependently H, phenyl, benzyl, linear or branched chain alkyl; whereinR″ is —CY═CHX, or H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein X is H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein nis 2 or 3; wherein R_(B) is hydrogen, t-butyloxycarbonyl,amyloxycarbonyl, (trialkylsilyl)alkyloxycarbonyl,(dialkylarylsilyl)alkyloxycarbonyl, benzyl, trialkylsilyl,dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl; and wherein R_(C)is tertiary-alkyl; which comprises reducing a hydroxy ketoesterdesoxyepothilone precursor having the structure:

[0204] wherein P, R, R₀, R_(B), R_(C), R′, R″ and n are defined asabove; under suitable conditions to form the hydroxy esterdesoxyepothilone precursor. In one embodiment, the invention providesthe method wherein n is 3 and R″ is 2-methyl-1,3-thiazolinyl. Inparticular, R_(A) is a trialkylsily group, and is, preferably, TES.R_(B) is Troc. In accord with the invention, the reducing step comprisescontacting the hydroxy ketoester desoxyepothilone precursor with astereospecific reducing reagent. The stereospecific reducing reagentfavorably comprises hydrogen gas at from about 900 pounds per squareinch to about 2200 pounds per square inch in the presence of(R)-(BINAP)RuCl₂ and optionally in the presence of HCl and an alcoholselected from the group consisting of MeOH, EtOH, and I-PrOH. Morepreferably, the hydrogen gas pressure is 1200 psi.

[0205] The present invention further provides a method of preparing ahydroxy ketoester desoxyepothilone precursor having the structure:

[0206] wherein P is H; wherein R, R₀, and R′ are independently H, linearor branched chain alkyl, optionally substituted by hydroxy, alkoxy,carboxy, carboxaldehyde, linear or branched alkyl or cyclic acetal,fluorine, NR₁R₂, N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ areindependently H, phenyl, benzyl, linear or branched chain alkyl; whereinR″ is —CY═CHX, or H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein X is H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein nis 2 or 3; wherein R_(B) is hydrogen, t-butyloxycarbonyl,amyloxycarbonyl, (trialkylsilyl)alkyloxycarbonyl, (dialkylarylsilyl)alkyloxycarbonyl, benzyl, trialkylsilyl, dialkylarylsilyl,alkyldiaryisilyl, triarysilyl, a linear or branched acyl, substituted orunsubstituted aroyl or benzoyl; and wherein R_(C) is tertiary-alkyl;which comprises deprotecting a protected ketoester desoxyepothiloneprecursor having the structure:

[0207] wherein R, R₀, R_(A), R_(B), R_(C), R′, R″ and n are defined asabove; and wherein P is a linear or branched alkyl, alkoxyalkyl,substituted or unsubstituted aryloxyalkyl, trialkylsilyl,aryldialkylsilyl, diarylalkylsilyl or triarylsilyl; under suitableconditions to form the hydroxy ketoester desoxyepothilone precursor. Inone embodiment, n is 3 and R″ is 2-methyl-1,3-thiazolinyl. R_(A) istypically trialkylsilyl, and, preferably, is TES. R_(B) is favorablyTroc. The method is effectively practiced wherein P is TBS. In accordwith the invention, the deprotecting step comprises contacting theprotected ketoester desoxyepothilone precursor with a protic acid.Preferably, the protic acid is HCl in methyl alcohol or ethyl alcohol.

[0208] The present invention also provides a method of preparing aprotected ketoester desoxyepothilone precursor having the structure:

[0209] wherein P is a linear or branched alkyl, alkoxyalkyl, substitutedor unsubstituted aryloxyalkyl, trialkylsilyl, aryldialkylsilyi,diarylalkylsilyl or triarylsilyl; wherein R, R₀, and R′ areindependently H, linear or branched chain alkyl, optionally substitutedby hydroxy, alkoxy, carboxy, carboxaldehyde, linear or branched alkyl orcyclic acetal, fluorine, NR₁R₂, N-hydroximino, or N-alkoxyimino, whereinR₁ and R₂ are independently H, phenyl, benzyl, linear or branched chainalkyl; wherein R″ is —CY═CHX, or H, linear or branched chain alkyl,phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl,2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl,3-indolyl or 6-indolyl; wherein X is H, linear or branched chain alkyl,phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl,2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl,3-indolyl or 6-indolyl; wherein Y is H or linear or branched chainalkyl; wherein n is 2 or 3; wherein R_(B) is hydrogen,t-butyloxycarbonyl, amyloxycarbonyl, (trialkylsilyl)alkyl-oxycarbonyl,(dialkylaryisilyl)alkyloxycarbonyl, benzyl, trialkylsilyl,dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, a linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl; and

[0210] wherein R_(C) is tertiary-alkyl; which comprises coupling aterminal vinyl enol ether ester having the structure:

[0211] wherein R, R₀, R_(B), R_(C), and R′ are defined as above; whereinm is 0, 1 or 2; and wherein R_(D) is linear or branched alkyl, benzyl,trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl; with a protectedhalovinyl or metalvinyl compound having the structure:

[0212] wherein R, P and R″ are defined as above; and wherein Q is ahalide or a metal; under suitable conditions to form the protectedketoester desoxyepothilone precursor. In one embodiment, the inventionprovides the method wherein n is 3 and R″ is 2-methyl-1,3-thiazolinyl.In another embodiment, the method is effectively performed wherein R_(A)is a trialkylsilyl group, and is, preferably, TES, and R_(B) is Troc. Pis favorably TBS or TES, and Q is iodine or bromine. R_(D) is typicallymethyl or TES. In accord with the invention, the coupling step comprisescontacting the terminal vinyl enol ether ester and the protectedhalovinyl compound with noble metal complex capable of effecting aSuzuki coupling. For this step, the noble metal complex is effectivelychosen as Pd(dppf)₂Cl₂ in the presence of Ph₃As and Cs₂CO₃.

[0213] The present invention also provides a method of preparing aterminal vinyl enol ether ester having the structure:

[0214] wherein R₀ and R′ are independently H, linear or branched chainalkyl, optionally substituted by hydroxy, alkoxy, carboxy,carboxaldehyde, linear or branched alkyl or cyclic acetal, fluorine,NR₁R₂, N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ areindependently H, phenyl, benzyl, linear or branched chain alkyl; whereinm is 0, 1 or 2; wherein R_(B) is hydrogen, t-butyloxycarbonyl,amyloxycarbonyl, (trialkylsilyl)alkyl-oxycarbonyl,(dialkylarylsilyl)alky-loxycarbonyl, benzyl, trialkylsilyl,dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl; wherein R_(C) istertiary-alkyl; and wherein R_(D) is linear or branched alkyl, benzyl,trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, linearor branched acyl, substituted or unsubstituted aroyl or benzoyl; whichcomprises:

[0215] (a) treating a keto enol ester having the structure:

[0216] under suitable conditions to form an enolate enol ester havingthe structure:

[0217] wherein M is Li, Na or K; and

[0218] (b) coupling the enolate enol ester with a vinyl aldehyde havingthe structure:

[0219] wherein m, and R₀ and R′ are as defined above; under suitableconditions to form the terminal vinyl enol ether ester. In accord withthe invention, the treating step comprises contacting the keto enolester with a strong nonnucleophilic base selected from the groupconsisting of lithium diethylamide, lithium diethylamide, lithiumdiisopropylamide, lithium hydride, sodium hydride, potassium hydride andpotassium t-butoxide. Preferably, the treating step is effected in apolar nonaqueous solvent selected from the group consisting oftetrahydrofuran, diethyl ether, di-n-propyl ether and dimethylformamideat a temperature from about −100° C. to about +10° C. More preferably,the temperature is from about −20° C. to −40° C. The coupling step aspracticed in the invention comprises contacting the enolate enol esterwith the vinyl aldehyde at a temperature from about −130° C. to about−78° C.

[0220] In addition, the invention provides a method of treating cancerin a subject suffering therefrom comprising administering to the subjecta therapeutically effective amount of any of the analogues related toepothilone B disclosed herein optionally in combination with anadditional chemotherapeutic agent and/or with a pharmaceuticallysuitable carrier. The method may be applied where the cancer is a solidtumor or leukemia. In particular, the method is applicable where thecancer is breast cancer or melanoma.

[0221] The subject invention also provides a pharmaceutical compositionfor treating cancer comprising any of the analogues of epothilonedisclosed hereinabove, as an active ingredient, optionally thoughtypically in combination with an additional chemotherapeutic agentand/or a pharmaceutically suitable carrier. The pharmaceuticalcompositions of the present invention may further comprise othertherapeutically active ingredients.

[0222] The subject invention further provides a method of treatingcancer in a subject suffering therefrom comprising administering to thesubject a therapeutically effective amount of any of the analogues ofepothilone disclosed hereinabove and a pharmaceutically suitablecarrier. The method is especially useful where the cancer is a solidtumor or leukemia.

[0223] The compounds taught above which are related to epothilones A andB are useful in the treatment of cancer, and particularly, in caseswhere multidrug resistance is present, both in vivo and in vitro. Theability of these compounds as non-substrates of MDR in cells, asdemonstrated in the Tables below, shows that the compounds are useful totreat, prevent or ameliorate cancer in subjects suffering therefrom.

[0224] The magnitude of the therapeutic dose of the compounds of theinvention will vary with the nature and severity of the condition to betreated and with the particular compound and its route ofadministration. In general, the daily dose range for anticancer activitylies in the range of 0.001 to 25 mg/kg of body weight in a mammal,preferably 0.001 to 10 mg/kg, and most preferably 0.001 to 1.0 mg/kg, insingle or multiple doses. In unusual cases, it may be necessary toadminister doses above 25 mg/kg.

[0225] Any suitable route of administration may be employed forproviding a mammal, especially a human, with an effective dosage of acompound disclosed herein. For example, oral, rectal, topical,parenteral, ocular, pulmonary, nasal, etc., routes may be employed.Dosage forms include tablets, troches, dispersions, suspensions,solutions, capsules, creams, ointments, aerosols, etc.

[0226] The compositions include compositions suitable for oral, rectal,topical (including transdermal devices, aerosols, creams, ointments,lotions and dusting powders), parenteral (including subcutaneous,intramuscular and intravenous), ocular (ophthalmic), pulmonary (nasal orbuccal inhalation) or nasal administration. Although the most suitableroute in any given case will depend largely on the nature and severityof the condition being treated and on the nature of the activeingredient. They may be conveniently presented in unit dosage form andprepared by any of the methods well known in the art of pharmacy.

[0227] In preparing oral dosage forms, any of the unusual pharmaceuticalmedia may be used, such as water, glycols, oils, alcohols, flavoringagents, preservatives, coloring agents, and the like in the case of oralliquid preparations (e.g., suspensions, elixers and solutions); orcarriers such as starches, sugars, microcrystalline cellulose, diluents,granulating agents, lubricants, binders, disintegrating agents, etc., inthe case of oral solid preparations are preferred over liquid oralpreparations such as powders, capsules and tablets. If desired, capsulesmay be coated by standard aqueous or non-aqueous techniques. In additionto the dosage forms described above, the compounds of the invention maybe administered by controlled release means and devices.

[0228] Pharmaceutical compositions of the present invention suitable fororal administration may be prepared as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the activeingredient in powder or granular form or as a solution or suspension inan aqueous or nonaqueous liquid or in an oil-in-water or water-in-oilemulsion. Such compositions may be prepared by any of the methods knownin the art of pharmacy. In general compositions are prepared byuniformly and intimately admixing the active ingredient with liquidcarriers, finely divided solid carriers, or both and then, if necessary,shaping the product into the desired form. For example, a tablet may beprepared by compression or molding, optionally with one or moreaccessory ingredients. Compressed tablets may be prepared by compressingin a suitable machine the active ingredient in a free-flowing form suchas powder or granule optionally mixed with a binder, lubricant, inertdiluent or surface active or dispersing agent. Molded tablets may bemade by molding in a suitable machine, a mixture of the powderedcompound moistened with an inert liquid diluent.

[0229] Methods of preparation of intermediates are disclosed in U.S.Patent Applications Serial Nos. 60/032,282, 60/033,767, 60/047,566,60/047,941, and 60/055,533, filed Dec. 3, 1996, Jan. 14, 1997, May 22,1997, May 29, 1997, and Aug. 13, 1997, respectively, the contents ofwhich are hereby incorporated by reference into this application.

[0230] The present invention will be better understood from theExperimental Details which follow. However, one skilled in the art willreadily appreciate that the specific methods and results discussed aremerely illustrative of the invention as described in the claims whichfollow thereafter. It will be understood that the processes of thepresent invention for preparing epothilones A and B, analogues thereofand intermediates thereto encompass the use of various alternateprotecting groups known in the art. Those protecting groups used in thedisclosure including the Examples below are merely illustrative.

EXAMPLE 1

[0231] THP Glycidol; 13:

[0232] A solution of (R)-(+)-glycidol 12 (20 g; 270 mmol) and freshlydistilled 3,4-dihydro-2H-pyran (68.1 g; 810 mmol) in CH₂Cl₂ (900 ml) wastreated with pyridinium p-toluenesulfonate (2.1 g; 8.36 mmol) at rt andthe resulting solution was stirred for 16 h. Approximately 50% of thesolvent was then removed in vacuo and the remaining solution was dilutedwith ether (1 L). The organic layer was then washed with two portions ofsaturated aqueous sodium bicarbonate (500 ml), dried (Na₂SO₄), filtered,and concentrated. Purification of the residue by flash chromatography(silica, 25→50% ether:hexanes) afforded THP glycidol 13 (31.2 g; 73%) asa colorless liquid: IR(film): 2941, 1122, 1034 cm⁻¹; ¹H NMR(CDCl₃, 500MHz) δ 4.66 (t, J=3.5 Hz, 1H), 4.64 (t, J=3.5 Hz, 1H), 3.93 (dd, J=11.7,3.1 Hz, 1H), 3.86 (m, 2H), 3.73 (dd, J=11.8, 5.03 Hz, 1H), 3.67 (dd,J=11.8, 3.4 Hz, 1H), 3.51 (m, 2H), 3.40 (dd, J=11.7, 6.4, 1H), 3.18 (m,2H), 2.80 (m, 2H), 2.67 (dd, J=5.2, 2.7 Hz, 1H), 2.58 (dd, J=5.0, 2.7Hz, 1H), 1.82 (m, 2H), 1.73 (m, 2H), 1.52 (m, 4H); ¹³C NMR (CDCl₃, 125MHz) δ 98.9, 98.8, 68.5, 67.3, 62.4, 62.2, 50.9, 50.6, 44.6, 44.5, 30.5,30.4, 25.4, 19.3, 19.2; [a]_(D)=+4.98 (c=2.15, CHCl₃).

EXAMPLE 2

[0233] Alcohol 13a:

[0234] Trimethylsilylacetylene (32.3 g; 329 mmol) was added via syringeto THF (290 ml), and the resulting solution was cooled to −78° C. andtreated with n-butyllithium (154 ml of a 1.6 M solution in hexanes;246.4 mmol). After 15 min, boron trifluoride diethyl etherate (34.9 g;246 mmol) was added, and the resulting mixture was stirred for 10 min. Asolution of epoxide 13 (26 g; 164.3 mmol) in THF (130 ml) was then addedvia a cannula and the resulting solution was stirred for 5.5 h at −78°C. The reaction was quenched by the addition of saturated aqueous sodiumbicarbonate solution (250 ml) and the solution was allowed to warm tort. The mixture was then diluted with ether (600 ml) and washedsuccessively with saturated aqueous sodium bicarbonate solution (250ml), water (250 ml), and brine (250 ml). The organic layer was thendried (Na₂SO₄), filtered, and concentrated in vacuo. Purification of theresidue by flash chromatography (silica, 20% ether:hexanes) providedalcohol 13a (34 g; 76%).

EXAMPLE 3

[0235] MOM ether 13b:

[0236] A solution of alcohol 13a (24 g; 88.9 mmol) andN,N-diisopropylethylamine (108 ml; 622 mmol) in anhydrous1,2-dichloroethane (600 ml) was treated with chloromethyl methyl ether(17 ml; 196 mmol), and the resulting mixture was heated to 55° C. for 28h. The dark mixture was then cooled to rt and treated with saturatedaqueous sodium bicarbonate solution (300 ml). The layers were separated,and the organic layer was washed successively with saturated aqueoussodium bicarbonate solution (200 ml) and brine (200 ml). The organiclayer was then dried (MgSO₄) and filtered through a pad of silica gel(ether rinse). Purification of the residue by flash chromatography(silica, 20→30% ether:hexanes) afforded MOM ether 13b (23.7 g; 85%) as apale yellow oil.

EXAMPLE 4

[0237] Alcohol 14:

[0238] A solution of THP ether 13b (20 g; 63.7 mmol) in methanol (90 ml)was treated with pyridinium p-toluenesulfonate (4.0 g; 15.9 mmol) andthe resulting mixture was stirred at rt for 16 h. The reaction was thenquenched by the addition of saturated aqueous sodium bicarbonatesolution (100 ml), and the excess methanol was removed in vacuo. Theresidue was diluted with ether (300 ml), and the organic layer waswashed successively with saturated aqueous sodium bicarbonate solution(200 ml) and brine (200 ml). The organic layer was dried (MgSO₄),filtered, and concentrated. Purification of the residue by flashchromatography (silica, 40→50% ether:hexanes) provided alcohol 14 (13.1g; 95%) as a colorless oil.

EXAMPLE 5

[0239] Alcohol 14a: To a cooled (−78° C.) solution of oxalyl chloride(24.04 ml of a 2.0 M solution in CH₂Cl₂; 48.08 mmol) in CH₂Cl₂ (165 ml)was added anhydrous DMSO (4.6 ml; 64.1 mmol) in dropwise fashion. After30 min, a solution of alcohol 14 (6.93 g; 32.05 mmol) in CH₂Cl₂ (65ml+10 ml rinse) was added and the resulting solution was stirred at −78°C. for 40 min. Freshly distilled triethylamine (13.4 ml; 96.15 mmol) wasthen added, the cooling bath was removed, and the mixture was allowed towarm to 0° C. The reaction mixture was then diluted with ether (500 ml),and washed successively with two portions of water (250 ml) and oneportion of brine (250 ml). The organic layer was then dried (MgSO₄),filtered, and concentrated.

[0240] The crude aldehyde (6.9 g) prepared in the above reaction wasdissolved in ether (160 ml) and cooled to 0° C. Methylmagnesium bromide(32.1 ml of a 3.0 M solution in butyl ether; 96.15 mmol) was then added,and the solution was allowed to warm slowly to rt. After 10 h, thereaction mixture was cooled to 0° C. and the reaction was quenched bythe addition of saturated aqueous ammonium chloride solution. Themixture was diluted with ether (200 ml) and washed successively withwater (150 ml) and brine (150 ml). The organic layer was dried (MgSO₄),filtered, and concentrated. Purification of the residue by flashchromatography (silica, 40→50% ether:hexanes) provided alcohol 14a (6.3g; 85% from 14).

EXAMPLE 6

[0241] Ketone 15:

[0242] A solution of alcohol 14 (1.0 g; 4.35 mmol), 4 A mol. sieves, andN-methylmorpholine-N-oxide (1.0 g; 8.7 mmol) in CH₂Cl₂ (20 ml) at rt wastreated with a catalytic amount of tetra-n-propylammonium perruthenate,and the resulting black suspension was stirred for 3 h. The reactionmixture was then filtered through a pad of silica gel (ether rinse), andthe filtrate was concentrated in vacuo. Purification of the residue byflash chromatography (silica, 10% ether:hexanes) afforded ketone 15 (924mg; 93%) as a light yellow oil.

EXAMPLE 7

[0243] Alkene 17:

[0244] A cooled (-78° C.) solution of phosphine oxide 16 (1.53 g; 4.88mmol) in THF (15.2 ml) was treated with n-butyllithium (1.79 ml of a2.45 M solution in hexanes). After 15 min, the orange solution wastreated with a solution of ketone 15 (557 mg; 2.44 mmol) in THF (4.6ml). After 10 min, the cooling bath was removed, and the solution wasallowed to warm to rt. The formation of a precipitate was observed asthe solution warmed. The reaction was quenched by the addition ofsaturated aqueous ammonium chloride solution (20 ml). The mixture wasthen poured into ether (150 ml) and washed successively with water (50ml) and brine (50 ml). The organic layer was dried (Na₂SO₄), filtered,and concentrated. Purification of the residue by flash chromatography(silica, 10% ether:hexanes) afforded alkene 17 (767 mg; 97%) as acolorless oil: IR(film): 2956, 2177, 1506, 1249, 1149, 1032, 842, cm⁻¹;¹H NMR(CDCl₃, 500 MHz) δ 6.95 (s, 1H), 6.53 (s, 1H), 4.67 (d, J=6.7 Hz,1H), 4.57 (d, J=6.8 Hz, 1H), 4.29 (dd, J=8.1, 5.4 Hz, 1H), 3.43 (s, 3H),2.70 (s, 3H), 2.62 (dd, J=16.9, 8.2 Hz, 1H), 2.51 (dd, J=17.0, 5.4 Hz,1H), 2.02 (s, 3H); ¹³C NMR (CDCl₃, 125 MHz) δ 164.4, 152.5, 137.1,121.8, 116.2, 103.7, 93.6, 86.1, 79.6, 55.4, 25.9, 19.1, 13.5;[α]_(D)=−27.3 (c=2.2, CHCl₃).

EXAMPLE 8

[0245] Alkynyl iodide formation:

[0246] To a solution of the alkyne 17 (3.00 g, 9.29 mmol) in acetone(100 mL) at 0° C. was added NIS (2.51 g; 11.2 mmol) and AgNO₃ (0.160 g;0.929 mmol). The mixture was then slowly warmed to rt. After 1.5 h, thereaction was poured into Et₂O (250 mL) and washed once with satbisulfite (40 mL), once with sat NaHCO₃ (40 mL), once with brine (40 mL)and dried over anhydrous MgSO₄. Purification by flash chromatography onsilica gel using gradient elution with hexanes/ethyl acetate (10:1-7:1)gave 2.22 g (64%) of the iodide 17a as an amber oil.

EXAMPLE 9

[0247] Reduction of the alkynyl iodide:

[0248] BH₃.DMS (0.846 mL, 8.92 mmol) was added to a solution ofcyclohexene (1.47 mL, 17.9 mmol) in Et₂O (60 mL) at 0° C. The reactionwas then warmed to rt. After 1 h, the iodide x (2.22 g, 5.95 mmol) wasadded to Et₂O. After 3 h, AcOH (1.0 mL) was added. After 30 additionalmin, the solution was poured into sat NaHCO₃ and extracted with Et₂O(3×100 mL). The combined organics were then washed once with brine (50mL) and dried over anhydrous MgSO₄. Purification by flash chromatographyon silica gel eluting with hexanes/ethyl acetate (6:1) gave 1.45 g (65%)of the vinyl iodide 18 as a yellow oil.

EXAMPLE 10

[0249] MOM removal:

[0250] To a solution of iodide 18 (1.45 g, 3.86 mmol) in CH₂Cl₂ (40 mL)at rt was added thiophenol (1.98 mL, 19.3 mmol) and BF₃oEt₂O (1.90 mL,15.43 mmol). After 22 h, the reaction was poured into EtOAc (150 mL) andwashed with 1 N NaOH (2×50 mL) and dried over anhydrous MgSo₄.Purification by flash chromatography on silica gel using gradientelution with hexanes/ethyl acetate (4:1-2:1-1:1) gave 1.075 g (86%) ofthe alcohol 18a as a pale yellow oil.

EXAMPLE 11

[0251] Acetate formation:

[0252] To a solution of alcohol 18a (1.04 g, 3.15 mmol) in CH₂Cl₂ (30mL) was added pyridine (2.52 mL, 25.4 mmol), acetic anhydride (1.19 mL,12.61 mmol) and DMAP (0.005 g). After 1 h, the volatiles were removed invacuo. Purification of the resulting residue by flash chromatography onsilica gel eluting with hexanes/ethyl acetate (7:1) gave 1.16 g (99%) ofthe acetate 19 as a pale yellow oil. IR(film):1737, 1368, 1232, 1018cm⁻¹; ₁H NMR (CDCl₃, 500 MHz) δ 6.97 (s, 1H), 6.53 (s, 1H), 6.34 (dd,J=17.5, 1.0 Hz, 1H), 6.18 (dd, J=13.7, 6.9 Hz, 1 h), 5.40 (t, J=6.4 Hz,1H), 2.70 (s, 3 h), 2.61 (m, 2H), 2.08 (2s, 6H). ¹³C NMR (CDCl₃, 125MHz) δ 169.8, 164.4, 152.2, 136.4, 136.1, 120.6, 116.4, 85.1, 38.3,21.0, 19.1, 14.7; [α]_(D)=−28.8 (c=1.47, CHCl₃).

EXAMPLE 12

[0253] To a solution of alcohol 4 (2.34 g, 3.62 mmol) and 2,6-lutidine(1.26 mL, 10.86 mmol) in CH₂Cl₂ (23 mL) at 0° C. was treated with TBSOTf(1.0 mL, 4.34 mmol). After stirrring for 1.5 h at 0° C. the reactionmixture was quenched with MeOH (200 uL) and the mixture stirred anadditional 5 min. The reaction mixture was diluted with Et₂O (100 mL)and washed successively with 1 N HCl (25 mL), water (25 mL), and brine(25 mL). The solution was dried over MgSO₄, filtered, and concentrated.The residue was purified by flash chromatography on silica gel elutingwith 5% Et₂O in hexanes to provide compund 7 (2.70 g, 98%) as acolorless foam.

EXAMPLE 13

[0254] A solution of compound 7 (2.93 g, 3.85 mmol) in CH₂Cl₂/H₂O (20:1,80 mL) was treated with DDQ (5.23 g, 23.07 mmol) and the resultingsuspension was stirred at room temperature for 24 h. The reactionmixture was diluted with Et₂O (200 mL) and washed with aqueous NaHCO₃(2×40 mL). The aqueous layer was extracted with Et₂O (3×40 mL) and thecombined organic fractions were washed with brine (50 mL), dried overMgSO₄, filtered, and concentrated. Purification of the crude oil byflash chromatography on silica gel eluting with 30% ether in hexanesafforded alcohol 7A (2.30 g, 89%) as a colorless oil: IR (film) 3488,1471, 1428, 1115, 1054 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 7.70 (6H, dd,J=8.0, 1.5 Hz), 7.44 (9H, s), 4.57 (1H, d, J=3.5 Hz), 4.19 (1H, s), 3.67(1H, d, J=8.5 Hz), 3.06 (1H, dd, J=11.5, 5.0 Hz), 2.89 (1H, dd, J=11.5,5.0 Hz), 2.68 (1H, d, J=13.5 Hz), 2.59 (1H, d, J=13.5 Hz), 2.34 (1H, dt,J=12.0, 2.5 Hz), 2.11 (1H, m), 1.84 (1H, dt, J=12.0, 2.5 Hz), 1.76 (2H,m), 1.59 (2H, m), 1.34 (3H, s), 1.13 (3H, d, J=7.5 Hz), 1.10 (3H, s),0.87 (9H, s), 0.84 (3H, d, J=12.0 Hz), 0.02 (3H, s), 0.01 (3H, s); ¹³CNMR (CDCl₃, 125 MHz) δ 136.18, 134.66, 130.16, 127.84, 78.41, 75.91,63.65, 59.69, 45.43, 45.09, 37.72, 30.84, 30.50, 26.23, 25.89, 22.42,21.05, 18.40, 15.60, 14.41, −3.23, −3.51; [α]_(D)=−0.95 (c=0.173,CHCl₃).

EXAMPLE 14

[0255] To a solution of oxalyl chloride (414 μL, 4.74 mmol) in CH₂Cl₂(40 mL) at −78° C. was added dropwise DMSO (448 uL, 6.32 mmol) and theresulting solution was stirred at −78° C. for 30 min. Alcohol 7a (2.12g, 3.16 mmol) in CH₂Cl₂ (20 mL) was added and the resulting whitesuspension was stirred at −78° C. for 45 min. The reaction mixture wasquenched with Et₃N (2.2 mL, 15.8 mmol) and the solution was allowed towarm to 0° C. and stirred at this temperature for 30 min. The reactionmixture was diluted with Et₂O (100 mL) and washed successively withaqueous NH₄Cl (20 mL), water (20 mL), and brine (20 mL). The crudealdehyde was purified by flash chromatography on silica gel eluting with5% Et₂O in hexanes to provide aldehyde 8 (1.90 g, 90%) as a colorlessoil.

EXAMPLE 15

[0256] A solution of (methoxymethyl)triphenylphosphonium chloride (2.97g, 8.55 mmol) in THF (25 mL) at 0° C. was treated with KOtBu (8.21 mL,1M in THF, 8.1 mmol). The mixture was stirred at 0° C. for 30 min.Aldehyde 8 (3.1 g, 4.07 mmol) in THF (10 mL) was added and the resultingsolution was allowed to warm to room temperature and stirred at thistemperature for 2 h. The reaction mixture was quenched with aqueousNH₄Cl (40 mL) and the resulting solution extracted with Et₂O (3×30 mL).The combined Et₂O fractions were washed with brine (20 ml), dried overMgSO₄, filtered, and concentrated. The residue was purified by flashchromatography on silica gel eluting with 5% Et₂O in hexanes to yieldcompound 9 (2.83 g, 86%) as a colorless foam.

EXAMPLE 16

[0257] To a solution of compound 9 (2.83 g, 3.50 mmol) in dioxane/H₂O(9:1, 28 mL) was added pTSA—H₂O (1.0 g, 5.30 mmol) and the resultingmixture was heated to 50° C. for 2 h. After cooling to room temperaturethe mixture was diluted with Et₂O (50 mL) and washed with aqueous NaHCO₃(15 mL), brine (20 ml), dried over MgSO₄, filtered, and concentrated toprovide aldehyde 9a (2.75 g, 99%) as a colorless foam.

EXAMPLE 17

[0258] Methyltriphenylphosphonium bromide (1.98 g, 5.54 mmol) in THF (50mL) at 0° C. was treated with lithium bis(trimethylsilyl)amide (5.04 mL,1M in THF, 5.04 mmol) and the resulting solution was stirred at 0° C.for 30 min. Aldehyde 9a (2.0 g, 2.52 mmol) in THF (5.0 mL) was added andthe mixture was allowed to warm to room temperature and stirred at thistemperature for 1 h. The reaction mixture was quenched with aqueousNH₄Cl (15 mL) and extracted with Et₂O (3×20 mL). The combined Et₂Ofractions were washed with brine (15 mL), dried over MgSO₄, filtered,and concentrated. The residue was purified by flash chromatography onsilica gel eluting with 5% Et₂O in hexanes to afford compound 10 (1.42g, 76%) as a colorless foam.

EXAMPLE 18

[0259] A solution of compound 10 (1.0 g, 1.34 mmol) in MeOH/THF (2:1, 13mL) was treated with [bis(trifluoroacetoxy)iodobenzene] (865 mg, 2.01mmol) at room temperature. After 15 min the reaction mixture wasquenched with aqueous NaHCO₃ (25 mL). The mixture was extracted withEt₂O (3×25 mL) and the combined Et₂O fractions were washed with brine,dried over MgSO₄, filtered, and concentrated. Purification of theresidue by flash chromatography on silica gel eluting with 5% Et₂O inhexanes provided compound 11 (865 mg, 92%) as a colorless foam: IR(film) 1428,1252,1114,1075,1046 cm¹; ¹H NMR (CDCl₃, 500 MHz) δ 7.61 (6H,dd, J=7.9, 1.4 Hz), 7.38 (9H, s), 5.47 (1H, m), 4.87 (1H, d, J=10.0 Hz),4.76 (1H, d, J=15.9 Hz), 4.30 (1H, d, J=3.7 Hz), 3.95 (1H, s), 3.56 (1H,dd, J=7.5, 1.4 Hz), 3.39 (3H, s), 2.84 (3H, s), 2.02 (1H, m), 1.64 (2H,m), 1.34 (1H, m), 1.11 (3H, s), 1.02 (3H, d, J=7.4 Hz), 0.90 (3H, s),0.85 (9H, s), 0.62 (3H, d, J=6.8 Hz), −0.04 (3H, s), −0.05 (3H, s); ¹³CNMR (CDCl₃, 125 MHz) δ 138.29, 135.79, 135.04, 129.86, 127.78, 114.98,110.49, 60.11, 55.57, 46.47, 43.91, 36.82, 34.21, 26.26, 19.60, 18.60,17.08, 16.16, 13.92, −2.96, −3.84; [α]_(D)=+1.74 (c=0.77, CHCl₃).

EXAMPLE 19

[0260] Suzuki Coupling:

[0261] To a solution of olefin 11 (0.680 g, 1.07 mmol) in THF (8.0 mL)was added 9-BBN (0.5 M soln in THF, 2.99 mL, 1.50 mmol). In a separateflask, the iodide 19 (0.478 g, 1.284 mmol) was dissolved in DMF (10.0mL). CsCO₃ (0.696 g, 2.14 mmol) was then added with vigorous stirringfollowed by sequential addition of Ph₃As (0.034 g, 0.111 mmol),PdCl₂(dppf)₂ (0.091 g, 0.111 mmol) and H₂O (0.693 mL, 38.5 mmol). After4 h, then borane solution was added to the iodide mixture in DMF. Thereaction quickly turned dark brown in color and slowly became paleyellow after 2 h. The reaction was then poured into H₂O (100 mL) andextracted with Et₂O (3×50 mL). The combined organics were washed withH₂O (2×50 mL), once with brine (50 mL) and dried over anhydrous MgSO₄.Purification by flash chromatography on silica gel eluting withhexanes/ethyl acetate (7:1) gave 0.630 g (75%) of the coupled product 20as a pale yellow oil.

EXAMPLE 20

[0262] Hydrolysis of Dimethyl Acetal 21:

[0263] The acetate 20 (0.610 g, 0.770 mmol) was dissolved in dioxane/H₂O(9:1,15 mL) and p-TSA.H 20 (0.442 g, 2.32 mmol) was added. The mixturewas then heated to 55° C. After 3 h, the mixture was cooled to rt andpoured into Et₂O. This solution was washed once with sat NaHCO₃ (30 mL),once with brine (30 mL) and dried over anhydrous MgSO₄. Purification byflash chromatography on silica gel eluting with hexanes/ethyl acetate(7:1) gave 0.486 g (85%) of the aldehyde 21 as a pale yellow oil. IR(film) 1737,1429,1237,1115, 1053 cm⁻¹; ¹H NMR(CDCl₃, 500 MHz) δ9.74 (1H,s), 7.61 (6H, dd, J=7.8, 1.2 Hz), 7.38 (9H, m), 6.94 (1H, s), 6.53 (1H,s), 5.39 (1H, m), 5.31 (1H, m), 5.29 (1H, t, J=6.9 Hz), 4.61 (1H, d,J=4.3 Hz), 3.50 (1H, dd, J=5.2, 2.6 Hz), 2.70 (3H, s), 2.48 (2H, m),2.14 (1H, m), 2.09 (3H, s), 2.07 (3H, s), 1.83 (2H, m), 1.41 (1H, m),1.18 (1H, m), 1.01 (3H, s), 0.99 (3H, s), 0.91 (3H, d, J=7.4 Hz), 0.85(9H, s), 0.69 (1H, m), 0.58 (3H, d, J=6.8 Hz), −0.05 (3H, s), −0.06 (3H,s); ¹³C NMR (CDCl₃, 125 MHz) δ 205.46, 170.01, 164.49, 152.46, 137.10,135.60, 134.22, 132.55, 130.65, 127.84, 123.82, 120.66, 116.19, 81.09,78.47, 76.73, 51.66, 43.14, 38.98, 30.99, 30.42, 27.63, 26.10, 21.15,20.92, 20.05, 19.15, 18.49, 15.12, 14.70, 12.75, −3.25, −4.08;[α]_(D)=−18.7 (c=0.53, CHCl₃).

EXAMPLE 21

[0264] Aldol:

[0265] To a solution of the acetate-aldehyde 21(84 mg,0.099 mmol) in THFat −78° C. was added KHMDS (0.5M in toluene, 1.0 ml, 0.5 mmol))dropwise. The resulting solution was stirred at −78° C. for 30 min. Thenthe reaction mixure was cannulated to a short pad of silica gel andwashed with ether. The residue was purified by flash chromatography(silica, 12% EtOAc in hexane) to give the lactone 22 (37 mg of 3-S and 6mg of 3-R, 51%) as white foam.

EXAMPLE 22

[0266] Monodeprotection:

[0267] Lactone 22 (32 mg, 0.0376 mmol) was treated with 1 ml of pyridinebuffered HF.pyridine—THF solution at room temperture for 2 h. Thereaction mixure was poured into saturated aqueous NaHCO₃ and extractedwith ether. The organic layer was washed in sequence with saturatedCUSO₄ (10 ml×3) and saturated NaHCO₃ (10 ml), then dried over Na₂SO₄ andconcentrated under vacuum. The residue was purified by flashchromatography (silica, 25% EtOAc in hexane) and to give diol 22a (22mg, 99%) as white foam.

EXAMPLE 23

[0268] TBS-Protection:

[0269] To a cooled (-30° C.) solution of diol 22a (29 mg, 0.0489 mmol)and 2,6-lutidine (0.017 ml, 0.147 mmol) in anhydrous CH₂Cl₂ (1 ml) wasadded TBSOTf (0.015 ml, 0.0646 mmol). The resulting solution was thenstirred at −30° C. for 30 min. The reaction was quenched with 0.5M HCl(10 ml) and extracted with ether (15 ml). Ether layer was washed withsaturated NaHCO₃, dried (Na₂SO₄) and concentrated in vacuo. Purifictionof the residue by flash chromatogrphy (silica, 8% EtOAc in hexane)afforded TBS ether 22B (32 mg, 93%) as white foam.

EXAMPLE 24

[0270] Ketone Formation:

[0271] To a solution of alcohol 22B (30 mg, 0.0424 mmol) in CH₂Cl₂ (2.0mL) at 25° C. was added Dess-Martin periodinane (36 mg, 0.0848 mmol) inone portion. The resulting solution was then allowed to stir at 25° C.for 1.5 h. The reaction was quenched by the addition of 1:1 saturatedaqueous sodium bicarbonate: sodium thiosulfate (10 ml) and stirred for 5min.

[0272] The mixture was then extracted with ether (3×15 ml). The organiclayer was dried (Na₂SO₄), filtered, and concentrated in vacuo.Purification of the residue by flash chromatography (silica, 8% EtOAc inhexane) provided ketone 22C (25 mg, 84%) as white foam. IR(film): 2928,1745,1692, 1254,1175,836 cm⁻¹; ¹H NMR(CDCl₃, 500 MHz) δ 6.97 (s, 1H),6.57 (s, 1H), 5.53 (dt, J=3.4, 11.1 Hz, 1H), 5.37 (dd, J=16.4, 9.9 Hz,1H), 5.00 (d, J=10.3 Hz, 1H), 4.02 (d, J=9.7 Hz, 1H), 3.89 (d, J=8.7 Hz,1H), 3.00 (m, 1H), 2.82 (d, J=6.5 Hz, 1H), 2.71 (m, 5H), 2.36 (q, J=10.7Hz, 1H), 2.12 (,3H), 2.07 (dd, J−8.2, 1H), 1.87 (bs, 1H), 1.49 (m, 3H),1.19 (m, 5H), 1.14 (s, 3H), 1.08 (d, J=6.8 Hz, 3H), 0.94 (m, 12H), 0.84(s, 9H), 0.12 (s, 3H), 0.10 (s, 3H), 0.07 (s, 3H), −0.098 (s, 3H); ¹³CNMR (CDCl₃, 125 MHz) δ 218.7, 170.1, 164.5, 152.6, 137.9, 133.9, 124.8,119.6, 115.9, 72.7, 53.2, 43.9, 41.0, 40.3, 32.9, 32.3, 28.4, 27.1,26.3, 26.1, 26.0, 19.2, 19.1, 18.3, 18.2, 17.1, 16.0, 15.2, 14.3, −4.2,−4.4, −4.6, 4.8 [α]_(D)=−21.93 (c=1.4, CHCl₃).

EXAMPLE 25

[0273] Desoxy Compound:

[0274] To a solution of TBS ether 22C (27 mg, 0.038 mmol) in THF(1 ml)at25° C. in a plastic vial was added dropwise HF.pyridine (0.5 ml). Theresulting solution was allowed to stir at 25° C. for 2 h. The reactionmixture was diluted with chloroform (2 ml) and very slowly added tosatured sodium bicarbonate (20 ml). The mixture was extracted with CHCl₃(20 ml×3). The organic layer was dried (Na₂SO₄), filtered, andconcentrated in vacuo. Purification of the residue by flashchromatography (silica, 30% EtOAc in hexane) provided diol 23 (18 mg,99%) as white foam: IR(film): 3493, 2925, 1728, 1689, 1249 cm⁻¹; ¹H NMR(CDCl₃, 500 MHz) δ 6.96 (s, 1H), 6.59 (s, 1H), 5.44 (dt, J=4.3, 10.4 Hz,1H), 5.36 (dt, J=5.1, 10.2 Hz, 1H), 5.28 (dd, J=1.7, 9.8 Hz, 1H), 4.11(d, J=7.2 Hz, 1H), 3.74 (s, 1H), 3.20 (d, J=4.5 Hz, 1H), 3.14 (dd,J=2.2, 6.8 Hz, 1H), 3.00 (s, 1H), 2.69 (m, 4H), 2.49 (dd, =11.3, 15.1Hz, 1H), 2.35 (dd, J−2.5, 15.1 Hz, 1H), 2.27 (m, 1H), 2.05 (m, 1H), 2.04(s, 3H), 2.01 (m, 1H) 1.75 (m, 1H), 1.67 (m, 1H), 1.33 (m, 4H), 1.21 (s,1H), 1.19 (m, 2H), 1.08 (d, J=7.0 Hz, 3H), 1.00 (s, 3H), 0.93 (d, J=7.1Hz, 3H); ¹³C NMR (CDCl₃, 125 MHz) δ 226.5, 176.5, 171.1, 158.2, 144.7,139.6, 131.1, 125.7, 122.0, 84.6, 80.2, 78.6, 59.4, 47.9, 45.4, 44.6,38.5, 37.9, 33.7, 33.6, 28.7, 25.1, 25.0, 21.9, 21.7, 19.6;[α]_(D)=−84.7 (c=0.85, CHCl₃).

EXAMPLE 26

[0275] Epothilone:

[0276] To a cooled (−50° C.) solution of desoxyepothilone (9 mg, 0.0189mmol) in dry CH₂Cl₂ (1 ml) was added freshly prepared dimethyldioxirane(0.95 ml, 0.1 M in acetone). The resulting solution was allowed to warmup to −30° C. for 2 h. A stream of nitrogen was then bubbled through thesolution to remove excess DMDO. The residue was purified by flashchromatography (silica, 40% EtOAc in hexane) and afforded epothilone A(4.6 mg, 49%) as colorless solid and 0.1 mg of cis-epoxide diastereomer.This material was identical with the natural epothilone A in allrespects.

EXAMPLE 27

[0277] Procedure for Ring-Closing Olefin Metathesis:

[0278] To a stirred solution of diene 24 (5 mg, 0.0068 mmol) in drybenzene (1.5 mL) was added Grubbs's catalyst (2.8 mg, 0.0034 mmol).After 12 h, an additional portion of catalyst was added (2.8 mg). Afteran additional 5 h, the reaction was concentrated. Purification by silicagel chromatography eluting with hexanes/ethyl acetate (11:1) gave thelactone 23 (3.5 mg, 94%, 2:1 E/Z).

EXAMPLE 28

[0279] Preparation of Compound 19:

[0280] Alcohol 2A:

[0281] A mixture of (S)-(−)-1,1′-bi-2-naphthol (259 mg. 0.91 mmol.),Ti(O-1-Pr)₄ (261 μL;0.90 mmol), and 4 A sieves (3.23 g) in CH₂C12 (16mL) was heated at reflux for 1 h. The mixture was cooled to rt andaldehyde 1 was added. After 10 min. the suspension was cooled to −78°C., and allyl tributyltin (3.6 mL; 11.60 mmol) was added. The reactionmixture was stirred for 10 min at −78° C. and then placed in a −20° C.freezer for 70 h. Saturated NaHCO₃ (2 mL) was added, and the mixture wasstirred for 1 h, poured over Na₂SO₄, and then filtered through a pad ofMgSO₄ and celite. The crude material was purified by flashchromatography (hexaneslethyl acetate, 1:1) to give alcohol 2A as ayellow oil (1.11 g; 60%).

EXAMPLE 29

[0282] Acetate 3A:

[0283] To a solution of alcohol 2A (264 mg; 1.26 mmol) in CH₂Cl₂ (12 mL)was added DMAP (15 mg: 0.098 mmol), Et₃N (0.45 mL; 3.22 mmol), and Ac₂O(0.18 mL; 1.90 mmol). After 2 h, the reaction mixture was quenched by 20mL of H₂O, and extracted with EtOAC (4×20 mL). The combined organiclayer was dried with MgSO₄, filtered, and concentrated. Flashchromatrography (EtOAC/hexanes, 1:3) afforded acetate 3A as a yellow oil(302 mg;

[0284] 96% ).

EXAMPLE 30

[0285] Vinyl Iodide 19:

[0286] To a solution of acetate 3A (99 mg; 0.39 mmol) in acetone at 0°C. was added H₂O (4 drops), OsO₄ (2.5% wt. in butyl alcohol; 175 μL;0.018 mmol), and N-methyl-morpholine-N-oxide (69 mg; 0.59 mmol). Themixture was stirred at 0° C. for 2 h and 45 min and then quenched withNa₂SO₃. The solution was poured to 10 mL of H₂O and extracted with EtOAc(5×10 mL). The combined organic layer was dried over MgSO4, filtered,and concentrated.

[0287] To a solution of this crude product in THF/H₂O (4 mL, 3:1) wasadded NaIO₄ (260 mg;

[0288] 1.22 mmol). After 1.25 h, the reaction mixture was then quenchedwith 10 mL of H₂O and concentrated. The residue was extracted with EtOAc(5×10 mL). The organic layer was dried over MgSO₄, filtered, andconcentrated. Flash chromatography (EtOAc/hexanes, 1:1) gave a yellowoil (80 mg) which contained unidentified by-product(s). This mixture wasused without further purification.

[0289] To a solution of (Ph₃P⁺CH₂I)I⁻ (100 mg; 0.19 mmol) in 0.25 mL ofTHF at rt was added 0.15 mL (0.15 mmol) of NaHMDS (1 M in THF). To theresulting solution at −78° C. was added HMPA (22 μL; 0.13 mmol) and theproduct from previous step (16 mg) in THF (0.25 mL). The reactionmixture was then stirred at rt for 30 min. After the addition of hexanes(10 mL), the solution was extracted with EtOAc (4×10 mL). The combinedEtOAC layer was dried (MgSO₄), filtered, and concentrated. PreparativeTLC (EtOAC/hexanes, 2.3) afforded vinyl iodide 19 as a yellow oil (14mg; 50% for three steps).

EXAMPLE 31

[0290] Iodoolefin Acetate 8C:

[0291] To a suspension of ethyltriphenylphosphonium iodide (1.125 g,2.69 mmol) in THF (10 mL) was added nBuLi (2.5 M soln in hexanes, 1.05mL, 2.62 mmol) at rt.

[0292] After disappearance of the solid material, the solution was addedto a mixture of iodine (0.613 g, 2.41 mmol) in THF (20 mL) at −78° C.The resulting suspension was vigorously stirred for 5 min at −78° C.,then warmed up −20° C., and treated with sodium hexamethyldisilazane (1M soln in THF, 2.4 mL, 2.4 mmol). The resulting red solution was stirredfor 5 min followed by the slow addition of aldehyde 9C (0.339 g, 1.34mmol). The mixture was stirred at −20° C. for 40 min, diluted withpentane (50 mL), filtered through a pad of celite, and concentrated.

[0293] Purification of the residue by flash column chromatography(hexanes/ethyl acetate, 85:15) gave 0.202 g (25% overall from vinylacetate 10C) of the vinyl iodide 8C as a yellow oil. IR (film): 2920,1738, 1234 cm⁻¹; ¹H NMR (CDCl₃): δ 6.98 (s, 1H), 6.56 (s, 1H), 5.42 (dd,J=5.43, 6.57 Hz, 1H), 5.35 (t, J=6.6 Hz, 1H), 2.71 (s, 3H), 2.54 (q,J=6.33, 2H), 2.50 (s, 3H), 2.09 (s, 6H); ¹³C NMR (CDCl₃): δ 170.1,164.6, 152.4, 136.9, 130.2, 120.6, 116.4, 103.6, 40.3, 33.7, 21.2, 19.2,14.9; [α]_(D)=−20.7° (c=2.45, CHCl₃).

EXAMPLE 32

[0294] Acetal 13C:

[0295] To a solution of olefin “7C” (0.082 g, 0.13 mmol) in THF (0.5 mL)was added 9-BBN (0.5 M soln in THF, 0.4 mL, 0.2 mmol). After stirring atrt. for 3.5 h, an additional portion of 9-BBN (0.5 M soln in THF, 0.26mL, 0.13 mmol) was added. In a separate flask, iodide BC (0.063 g, 0.16mmol) was dissolved in DMF (0.5 mL). Cs₂CO₃ (0.097 g, 0.30 mmol) wasthen added with vigorous stirring followed by sequential addition ofPdCl₂(dppf)₂ (0.018 g, 0.022 mmol), Ph₃As (0.0059 g, 0.019 mmol), andH₂O (0.035 mL, 1.94 mmol). After 6 h, then borane solution was added tothe iodide mixture in DMF. The reaction quickly turned dark brown incolor and slowly became pale yellow after 3 h. The reaction was thenpoured into H₂O (10 mL) and extracted with Et₂O (3×15 mL). The combinedorganic layers were washed with H₂O (3×15 mL), brine (1×20 mL), driedover MgSO₄, filtered, and concentrated. Flash column chromatography(hexanes/ethyl acetate, 9:1) gave 0.089 g (77%) of the coupled product13C as a yellow oil.

EXAMPLE 33

[0296] Aldehyde 14C:

[0297] Acetal 13C (0.069 g, 0.077 mmol) was dissolved in dioxane/H₂O(9:1, 1 mL) and pTSA.H₂O (0.045 g, 0.237 mmol) was added. The mixturewas then heated to 55° C.

[0298] After 3 h, the mixture was cooled to rt, poured into Et₂O, andextracted with Et₂O (4×15 mL).

[0299] The combined ether solutions were washed with sat NaHCO₃ (1×30mL), brine (1×30 mL), dried over MgSO₄, filtered, and concentrated.Flash column chromatography (hexanes/ethyl acetate, 3:1) gave 0.046 g(71%) of the aldehyde 14C as a pale yellow oil.

EXAMPLE 34

[0300] Macrocycle 15C-(SR):

[0301] To a solution of aldehyde 14C (0.021 g, 0.024 mmol) in THF (5 mL)at −78° C. was added KHMDS (0.5 M soln in toluene, 0.145 mL, 0.073mmol). The solution was stirred at −78° C. for 1 h, then quenched withsat'd NH₄Cl, and extracted with ether (3×15 mL). The combined organiclayers were dried with MgSO₄, filtered, and concentrated.

[0302] Flash column chromatography (hexanes/ethyl acetate, 7:1) gave0.008 g of the desired α-alcohol 15C—(S) and 0.006 g of β-alcohol15C—(R) (67% total ) as pale yellow oils.

EXAMPLE 35

[0303] Macrocycle 15C—(S):

[0304] To a solution of β-alcohol 15C—(R) (0.006 g, 0.0070 mmol) in 0.5mL of CH₂Cl₂ at rt. was added Dess-Martin periodinane (0.028 g, 0.066mmol). After 0.5 h, an additional portion of Dess-Martin periodinane(0.025 mg, 0.059 mmol) was added. The resulting solution was stirred atrt for additional 1 h, then treated with ether (2 mL) and sat'dNa₂S₂O₃/sat'd NaHCO₃,(3 mL, 1:1), poured into H₂O (20 mL), and extractedwith ether (4×10 mL). The combined ether solutions were washed with H₂O(1×30 mL), brine (1×30 mL), dried with MgSO₄, filtered, andconcentrated. To a solution of crude ketone 15C′ in MeOH/THF (2 mL, 1:1)at −78° C. was added NaBH₄ (0.015 g, 0.395 mmol). The resulting solutionwas stirred at rt for 1 h, quenched with sat NH₄Cl, and extracted withether (3×15 mL). The organic layers were dried with MgSO₄, filtered, andconcentrated. Flash column chromatography (hexanes/ethyl acetate, 9:1)gave 0.0040 g (67%) of the α-alcohol 15C—(S) as a pale yellow oil and0.0006 g of β-alcohol 15C—(R).

EXAMPLE 36

[0305] Diol 15C″:

[0306] The silyl ether 15C—(S) (0.010 g, 0.012 mmol) was dissolved inHF.pyridine/pyridine/THF (1 mL). The solution was stirred at rt. for 2h, then diluted with Et₂O (1 mL), poured into a mixture of Et₂O/sat.NaHCO₃ (20 mL, 1:1), and extracted with Et₂O (4×10 mL). The Et₂Osolutions were washed with sat CuSO₄ (3×30 mL), sat NaHCO₃ (1×30 mL),brine (1×30 mL), dried with MgSO₄, filtered, and concentrated. Flashcolumn chromatography (hexanes/ethyl acetate, 9:1) gave 0.0066 g (93%)of the diol 15C″ as a pale yellow oil.

EXAMPLE 37

[0307] Alcohol 15C′″:

[0308] To a solution of diol 15C″ (0.0066 g, 0.011 mmol) in 0.5 mL ofCH₂Cl₂ at-78 ° C. was added 2,6-lutidine (7 μL, 0.060 mmol) and TBSOTf(5 pL, 0.022 mmol). The resulting solution was stirred at −30° C. for0.5 h, then quenched with H₂O (5 mL), and extracted with Et₂O (4×10 mL).The ether solutions were washed with 0.5 M HCl (1×10 mL), sat'd NaHCO₃(1×10 mL), dried over MgSO₄, filtered, and concentrated. Flash columnchromatography (hexanes/ethyl acetate, 93:7) gave 0.0070 g (89%) of thealcohol 15C′″ as a pale yellow oil.

EXAMPLE 38

[0309] Ketone 16C:

[0310] To a solution of alcohol 15C′″ (0.006 g, 0.0083 mmol) in 0.5 mLof CH₂Cl₂ at rt. was added Dess-Martin periodinane (0.030 g, 0.071mmol). After 1.25 h, another portion of Dess-Martin periodinane (0.025mg, 0.059 mmol) was added. The resulting solution was stirred at rt foradditional 0.75 h, treated with ether (1 mL) and sat'd Na₂S₂O₃/sat'dNaHCO₃ (2 mL, 1:1), poured into H₂O (20 mL), and extracted with ether(4×10 mL). The ether solution was washed with sat NaHCO₃ (1×20 mL),dried with MgSO₄, filtered, and concentrated. Flash columnchromatography (hexanes/ethyl acetate, 9:1) gave 0.0040 g (67%) of theketone 16C as a pale yellow oil.

EXAMPLE 39

[0311] Desoxyepothiolone B 2C:

[0312] To a solution of ketone 16C (0.004 g, 0.0056 mmol) in THF (0.35mL) was added HF-pyridine (0.25 mL) dropwise over 20 min. The solutionwas stirred at rt for 1.5 h, diluted with CHCl₃ (2 mL), poured intosat'd NaHCO₃/CHCl₃ (20 mL, 1:1) slowly, and extracted with CHCl₃ (4×10mL). The combined CHCl₃ layers were dried with MgSO₄, filtered, andconcentrated. Flash column chromatography (hexaneslethyl acetate, 3:1)gave 0.0022 g (80%) of the desoxyepothilone B 2C as a pale yellow oil.

EXAMPLE 40

[0313] Epothilone B 2:

[0314] To a solution of desoxyepothilone B (0.0022 g, 0.0041 mmol) inCH₂Cl₂ (0.25 mL) at −50° C. was added dimethyldioxirane (0.1 mL, 0.0095mmol) dropwise. The resulting solution was stirred at −50° C. for 1 h.The dimethyldioxirane and solvent were removed by a stream of N₂. Theresidue was purified by flash column chromatography (hexaneslethylacetate, 1:1) gave 0.0015 g (70%) of epothiolone B (2) as a pale yellowoil which was identical with an authentic sample in ¹H NMR, IR, massspectrum, and [α]_(D).

EXAMPLE 41

[0315] 8-Desmethylepothilone A

[0316] Crotylation product:

[0317] To a stirred mixture of potassium tert-butoxide (1.0 M soln inTHF, 50.4 mL, 50.4 mmol), THF (14 mL), and cis-2-butene (9.0 mL, 101mmol) at −78° C. was added n-BuLi (1.6 M, in hexanes, 31.5 mL, 50.4mmol). After complete addition of n-BuLi, the mixture was stirred at−45° C. for 10 min and then cooled to −78° C.(+)-B-Methoxydiisopinocampheylborane (19.21 g, 60.74 mmol) was thenadded dropwise in Et₂O (10 mL). After 30 min, BF₃ Et₂O (7.47 mL, 60.74mmol) was added followed by aldehyde 4D (9.84 g, 60.74 mmol) in THF (15mL) generating a viscous solution which could not be stirred.

[0318] The mixture was shaken vigorously every 10 min to ensurehomogeneity. After 3 h at −78° C., the reaction was treated with 3N NaOH(36.6 mL, 110 mmol) and 30% H₂O₂ (15 mL) and the solution brought toreflux for 1 h. The reaction was poured into Et₂O (300 mL) and washedwith H₂O (100 mL), brine (30 mL) and dried over anhydrous MgSO₄. Thecrude material was placed in a bulb-to-bulb distillation apparatus toremove the ligand from the desired product.

[0319] Heating at 80° C. at 2 mm Hg removed 90% of the lower boilingligand. Further purification of the alcohol 4D was achieved by flashchromatography on silica gel eluting with Et₂O in CH₂Cl₂ (2%→4%) to givepure alcohol 4D as a clear oil. The erythro selectivty was >50:1 asjudged by ¹H NMR spectroscopy. The product was determined to be 87% eeby formation of the Mosher ester: IR (film): 3435, 2861, 1454, 1363,1099 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 7.34 (5H, m), 5.80 (1H, m), 5.09(1H, dd, J 1.6, 8.3 Hz), 5.04 (1H, d, J=1.6 Hz), 4.52 (2H, s), 3.51 (2H,t, J=5.8 Hz), 3.47 (1H, m), 2.27 (2H, m), 1.73 (3H, m), 1.42 (1H, m),1.04 (3H, d, J=6.9 Hz); ¹³C NMR (CDCl₃, 100 MHz) δ 141.1, 138.2, 128.3,127.6, 127.5, 115.0, 74.5, 72.9, 70.4, 43.7, 31.3, 26.5, 14.6.

EXAMPLE 42

[0320] TBS Ether 5D:

[0321] Alcohol 4D (5.00 g, 21.4 mmol) was dissolved in CH₂Cl₂ (150 mL)and 2,6-lutidine (9.97 mL, 85.6 mmol) was added. The mixture was cooledto 0° C. and TBSOTf (9.83 mL, 42.8 mmol) was slowly added. The reactionwas then warmed to rt. After 1 h, the reaction was poured into Et₂O (300mL) and washed once with 1 N HCl (50 mL), once with sat NaHCO₃ (50 mL),once with brine (30 mL) and dried over anhydrous MgSO₄. Purification byflash chromatography on silica gel eluting with hexanes/diethyl ether(97:3) gave 6.33 g (85%) of pure olefin 5D as a clear oil: IR (film):1472, 1361, 1255,1097, 1068 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 7.30 (5H,m), 5.81 (1H, m), 4.97 (1H, dd, J=1.4, 4.8 Hz), 4.94 (1H, d, J=1.1 Hz),3.51 (1H, q, J=5.1 Hz), 3.41 (2H, dt, J=2.1, 6.6 Hz), 2.27 (1H, q, J=5.5Hz), 1.68 (1 h, m), 1.55 (1H, m), 1.41 (2H, m), 0.93 (3H, d, J=6.9 Hz),0.85 (9H, s), −0.01 (6H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 141.2, 138.6,128.3, 127.6, 127.4, 113.9, 75.6, 72.7, 70.6, 42.7, 30.1, 25.9, 25.4,18.1, 15.1, −4.3, −4.4.

EXAMPLE 43

[0322] Aldehyde 6D:

[0323] The olefin 5 (4.00 g, 11.49 mmol) was dissolved in 1:1MeOH/CH₂Cl₂ (100 mL). Pyridine (4.0 mL) was then added and the mixturecooled to −78° C. Ozone was then bubbled through the reaction for 10minutes before the color turned light blue in color. Oxygen was thenbubbled through the reaction for 10 min. Dimethyl sulfide (4.0 mL) wasthen added and the reaction slowly warmed to rt. The reaction wasstirred overnight and then the volatiles were removed in vacuo.Purification by flash chromatography on silica gel eluting withhexanes/ethyl acetate (9:1) gave 3.31 g (82%) of the aldehyde 6D as aclear oil: IR (film): 2856, 1727, 1475, 1361, 1253, 1102 cm⁻¹; ¹H NMR(CDCl₃, 400 MHz) δ 9.76 (1 H, s), 7.33 (5H, m), 4.50 (2H, s), 4.11 (1H,m), 3.47 (2H, m), 2.46 (1H, m), 1.50-1.70 (4H, band), 1.05 (3H, d, J=7.0Hz), 0.86 (9H, s), 0.06 (3H, s), 0.03 (3H, s); ¹³C NMR (CDCl₃, 100 MHz)δ 204.8, 138.3, 128.2, 127.4, 127.3, 72.7, 71.7, 69.9, 51.1, 31.1, 25.9,25.6, 17.8, 7.5, −4.4, −4.8.

EXAMPLE 44

[0324] Dianion Addition Product 7D:

[0325] The tert-butyl isobutyrylacetate (0.653 g, 3.51 mmol) was addedto a suspension of NaH (60% in mineral oil, 0.188 g, 4.69 mmol) in THF(50 mL) at rt. After 10 min, the mixture was cooled to 0° C. After anadditional 10 min, n-BuLi (1.6 M in hexanes, 2.20 mL, 3.52 mmol) wasslowly added. After 30 min, the aldehyde 6D (1.03 g, 2.93 mmol) wasadded neat. After 10 min, the reaction was quenched with H₂O (10 mL) andextracted with Et₂O (2×75 mL). The combined organics were washed oncewith brine (30 mL) and dried over anhydrous MgSO₄. The crude reactionmixture contained a 15:1 ratio of diastereomers at C5. Purification byflash chromatography on silica gel eluting with hexanes/ethyl acetate(9:1→7:1) gave 0.723 g (47%) of the desired alcohol 7D as a clear oil:

[0326] IR (film): 3531, 2953, 1739, 1702, 1367, 1255, 1153 cm⁻¹; ¹H NMR(CDCl₃, 400 MHz) δ 7.33 (5H, m), 4.49 (2H, s), 3.75 (1H, d, J=2.6 Hz),3.71 (1H, m), 3.62 (1H, d, J=16.0 Hz), 3.53 (1H, d, J=16.0 Hz), 3.44(2H, t, J=5.1 Hz), 2.70 (1H, d, J=2.6 Hz), 1.83 (1 H, m), 1.55 (4H, m),1.46 (9H, s), 1.17 (3H, s), 1.11 (3H, s), 0.89 (9H, s), 0.82 (3H, d,J=7.0 Hz), 0.09 (6H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 208.9, 167.3, 138.4,128.3, 127.6, 127.5, 81.3, 79.5, 78.7, 72.8, 70.1, 52.4, 47.6, 35.8,30.6, 28.2, 25.9, 25.8, 22.6, 20.5, 17.9, 7.05, −4.0, −4.5.

EXAMPLE 45

[0327] Directed Reduction:

[0328] To a solution of tetramethylammonium triacetoxyborohydride (1.54g, 5.88 mmol) in acetonitrile (4.0 mL) was added anhydrous AcOH (4.0mL). The mixture was stirred at rt for 30 min before cooling to −10C. Asolution of the ester 7D (0.200 g, 0.39 mmol) in acetonitrile (1.0 mL)was added to the reaction and it was stirred at −10° C. for 20 h.

[0329] The reaction was quenched with 1 N sodium-potassium tartrate (10mL) and stirred at rt for 10 min. The solution was then poured into satNaHCO₃ (25 mL) and neutralized by the addition of solid Na₂CO₃. Themixture was then extracted with EtOAc (3×30 mL) and the organics werewashed with brine (20 mL) and dried over anydrous MgSO₄. Purification byflash chromatography on silica gel eluting with hexanes/ethyl acetate(4:1) gave 0.100 g (50%) of the diol as 10:1 ratio of diastereomericalcohols.

EXAMPLE 46

[0330] Monoprotection of the Diol:

[0331] The diol (1.76 g, 3.31 mmol) was dissolved in CH₂Cl₂ (100 mL) andcooled to 0° C. 2,6-lutidine (12.2 mL, 9.92 mmol) was added followed byTBSOTf (1.14 mL, 4.96 mmol) and the reaction slowly warmed to rt. After1 h, the reaction was poured into Et₂O (300 mL) and washed once with 1 NHCl (50 mL), once with sat NaHCO₃ (50 mL), once with brine (30 mL) anddried over anhydrous MgSO₄. Purification by flash chromatography onsilica gel eluting with hexanes/ethyl acetate (20:1→15:1) gave 2.03 g(95%) of the alcohol 8D as a clear oil, which was used as a mixture ofdiastereomers.

EXAMPLE 47

[0332] C5 Ketone Formation:

[0333] The alcohol 8D (2.03 g, 3.14 mmol) was dissolved in CH₂Cl₂ (50mL) and Dess-Martin periodinane (2.66 g, 6.28 mmol) was added. After 2h, a 1:1 mixture of sat'd NaHCO₃/sat Na₂S₂O₃ (20 mL) was added. After 10min, the mixture was poured into Et₂O (300 mL) and the organic layer waswashed with brine (30 mL) and dried over anhydrous MgSO₄. Purificationby flash chromatography on silica gel eluting with hexanes/ethyl acetate(15:1) gave 1.85 g (91%) of the ketone (benzyl ether) as a clear oil,which was used as a mixture of diastereomers.

EXAMPLE 48

[0334] Debenzylation:

[0335] The ketone (benzyl ether) (1.85 g, 2.87 mmol) was dissolved inEtOH (50 mL), and Pd(OH)₂ (0.5 g) was added. The mixture was thenstirred under an atmosphere of H₂. After 3 h, the reaction was purgedwith N₂ and then filtered through a pad of celite rinsing with CHCl₃(100 mL). Purification by flash chromatography on silica gel elutingwith ethyl acetate in hexanes (12→5%) gave 1.43 g (90%) of thediastereomeric alcohols as a clear oil. The C3 diastereomers wereseparated by flash chromatography on TLC-grade SiO2 eluting with ethylacetate in hexanes (15%):

[0336] Alpha isomer: IR (film): 3447, 1732, 1695, 1254, 1156 cm⁻¹; ¹HNMR (CDCl₃, 400 MHz) δ 4.24 (1H, dd, J=3.6, 5.8 Hz), 3.83 (1H, m), 3.53(1H, m), 3.06 (1H, t, J=7.1 Hz), 2.36 (1H, dd, J=3.6, 17.2 Hz), 2.12(1H, dd, J=3.9, 17.2 Hz), 1.68 (1H, t, J=5.4 Hz), 1.54 (2H, m), 1.41(1H, m), 1.37 (9H, s), 1.31 (1H, m), 1.16 (3H, s), 1.02 (3H, s), 0.99(3H, d, J=6.8 Hz), 0.84 (9H, s), 0.81 (9H, s), 0.05 (3H, s), 0.01 (6H,s), −0.01 (3H, s); ¹³C NMR(CDCl₃, 100 MHz) δ 217.7, 171.3, 80.57, 73.5,73.1, 63.0, 53.4, 26.8, 41.2, 32.1, 28.1, 28.0, 26.0, 25.9, 23.1, 19.8,18.1 (overlapping), 15.3, −4.0, −4.3 (overlapping), −4.8.

[0337] Beta isomer: IR (film): 3442, 2857, 1732, 1700, 1472, 1368, 1255cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 4.45 (1H, t, J=5.3 Hz), 3.86 (1H, m),3.52 (2H, q, J=5.9 Hz), 3.01 (1H, m), 2.28 (1H, dd, J=4.3, 17.1 Hz),2.16 (1H, dd, J=5.5, 17.1 Hz), 1.67 (1H, t, J=5.6 Hz), 1.56 (2H, m),1.44 (1H, m), 1.37 (9H, s), 1.34 (1H, m), 1.13 (3H, s), 0.97 (3H, d,J=7.4 Hz), 0.96 (3H, s), 0.83 (9H, s), 0.79 (9H, s), 0.01 (3H, s), 0.00(6H, s), −0.07 (3H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 217.1, 171.2, 80.6,73.5, 72.1, 62.9, 63.9, 46.4, 41.2, 32.0, 28.1, 28.0, 26.0, 25.9, 21.5,19.5, 18.2, 18.1, 15.8, −4.0, −4.3, −4.4, −4.7.

EXAMPLE 49

[0338] Aldehyde Formation:

[0339] DMSO (0.177 mL,2.50 mmol) was added to a mixture of oxalylchloride (0.11 mL, 1.25 mmol) in CH₂Cl₂ (15 mL) at −78° C. After 10 min,the alcohol (0.531 g, 0.96 mmol) was added in CH₂Cl₂ (4 mL). After 20min, TEA (0.697 mL, 5.00 mmol) was added to the reaction followed bywarming to rt. The reaction was then poured into H₂O (50 mL) andextracted with Et₂O (3×50 mL). The organics were washed once with H₂O(30 mL), once with brine (30 mL) and dried over anhydrous MgSO₄. Thealdehyde was used in crude form.

EXAMPLE 50

[0340] Wittig Olefination to give 9D:

[0341] NaHMDS (1.0 M soln in THF, 1.54 mL, 1.54 mmol) was added to asuspension of methyl triphenylphosphonium bromide (0.690 g, 1.92 mmol)in THF (20 mL) at 0° C. After 1 h, the crude aldhyde (0.96 mmol) wasadded in THF (5 mL). After 15 min at 0° C., H₂O (0.1 mL) was added andthe reaction poured into hexanes (50 mL). This was filtered through aplug of silica gel eluting with hexanes/Et₂O (9:1, 150 mL). The crudeolefin 9D was further purified by flash chromatography on silica geleluting with ethyl acetate in hexanes (5%) to give 0.437 g (83% for twosteps) of the olefin 9D as a clear oil: IR (film): 2857, 1732, 1695,1472, 1368, 1255, 1156 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 5.72 (1H, m),4.91 (2H, m), 4.25 (1H, dd, J=3.9, 5.4 Hz), 3.81 (1H, m), 3.05 (1H, m),2.38 (1H, dd, J=7.9, 17.2 Hz), 2.12 (1H, dd, J=6.6, 17.2 Hz), 2.04 (2H,q, J=7.5 Hz), 1.47 (1H, m), 1.39 (9H, s), 1.34 (1H, m), 1.20 (3H, s),1.00 (3H, s), 3.00 (3H, d, J=6.7 Hz), 0.85 (9H, s), 0.83 (9H, s), 0.07(3H, s), 0.00 (6H, s), −0.05 (3H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 217.5,172.1, 137.9, 114.0, 80.4, 74.0, 73.0, 53.0, 46.9, 41.3, 35.1, 29.0,28.1, 26.0, 25.9, 22.8, 20.2, 18.2 (overlapping), 14.9, −4.1, −4.2,−4.3, −4.8.

EXAMPLE 51

[0342] TBS Ester 10D:

[0343] The olefin 9D (0.420 g, 0.76 mmol) was dissolved in CH₂Cl₂ (15mL) and treated successively with 2,6-lutidine (1.33 mL, 11.4 mmol) andTBSOTf (1.32 mL, 5.73 mmol). After 7 h, the reaction was poured intoEt₂O (100 mL) and washed successively with 0.2N HCl (25 mL), brine (20mL) and dried over anhydrous MgSO₄. The residue was purified by flashchromatography on a short pad of silica gel with fast elution withhexanes/ethyl acetate (20:1) to give the TBS ester 10D as a clear oil.The purification must be done quickly to avoid hydrolysis of the silylester: IR (film): 2930, 1721, 1695, 1472, 1254, 1091 cm⁻¹;

[0344]¹H NMR (CDCl₃, 400 MHz) δ 5.73 (1H, m), 4.91 (2H, m), 4.25 (1H,dd, J=3.8, 5.4 Hz) 3.80 (1H, q, J=6.8 Hz), 3.06 (1H, m), 2.50 (1H, dd,J=3.7, 17.3 Hz), 2.19 (1H, dd, J=5.7, 17.3 Hz), 2.04 (2H, dd, J=7.6,15.3 Hz), 1.49 (1H, m), 1.36 (1H, m), 1.21 (3H, s), 1.00 (3H, d, J=6.8Hz), 0.88 (9H, s), 0.85 (9H, s), 0.83 (9H, s), 0.22 (3H, s), 0.22 (3H,s), 0.21 (3H, s), 0.06 (3H, s), 0.01 (6H, s), −0.05 (3H, s); ¹³C NMR(CDCl₃, 100 MHz) δ 217.3, 172.3, 138.5, 114.4, 74.5, 73.0, 53.2, 46.9,41.8, 35.1, 29.0, 26.0, 25.7, 25.5, 22.8, 20.4, 18.2, 18.1, 17.5, 14.9,−2.9, −4.0, −4.2, −4.3, −4.8, −4.9.

EXAMPLE 52

[0345] Suzuki Coupling:

[0346] The acetate acid 13D was purified by flash chromatography onsilica gel eluting with hexanes/ethyl acetate (7:1-4:1). This wasfurther purified by preparative-TLC eluting with hexanes/ethyl acetate(2:1) to remove unreacted vinyl iodide 12D from the acetate acid 13D.Isolated yield of the acid was 0.297 g (62% based on 90% purity withborane residues).

EXAMPLE 53

[0347] Hydrolvsis of Acetate Acid 13D:

[0348] The acetate 13D (0.220 g, 0.297 mmol) was dissolved in MeOH/H₂O(2:1, 15 mL) and K₂CO₃ (0.300 g) was added. After 3 h, the reaction wasdiluted with sat NH₄Cl (20 mL) and extracted with CHCl₃ (5×20 mL). Thehydroxy-acid 14D was purified by flash chromatography on silica geleluting with hexanes/ethyl acetate (4:1→2:1) to give 0.146 g (70%) ofthe pure hydroxy acid 14D. IR (film): 3510-2400, 1712, 1694, 1471, 1254,1093 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 6.96 (1H, s), 6.66 (1H, s), 5.55(1H, m), 5.38 (1H, m), 4.38 (1H, dd, J=3.4, 6.1 Hz), 4.19 (1H, t, J=7.5Hz), 3.84 (1H, m), 3.05 (1H, t, J=7.0 Hz), 2.72 (3H, s), 2.49 (1H, dd,J=3.2, 16.4 Hz), 2.42 (2H, m), 2.33 (1H, dd, J=6.2, 16.4 Hz), 2.07 (2H,m), 2.02 (3H, s), 1.33 (4H, m), 1.19 (3H, s), 1.14 (3H, s), 1.06 (3H, d,J=6.7 Hz), 0.89 (9H, s), 0.88 (9H, s), 0.11 (3H, s), 0.07 (3H, s), 0.04(6H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 217.8, 176.6, 164.9, 152.5, 141.7,132.9, 125.0, 119.0, 115.3, 73.5, 73.3, 53.4, 47.0, 40.1, 35.8, 33.2,29.8, 27.4, 26.0, 25.9, 24.5, 19.0, 18.1, 15.2, 14.3, −4.0, −4.2, −4.2,−4.7.

EXAMPLE 54

[0349] Macrolactonization:

[0350] DCC (0.150 g, 0.725 mmol), 4-DMAP (0.078 g, 0.64 mmol) and4-DMAP.HCl (0.110 g, 0.696 mmol) were dissolved in CHCl₃ (80 mL) at 80°C. To this refluxing solution was added by syringe pump the hydroxy acid14D (0.020 g, 0.029 mmol) and DMAP (0.010 g) in CHCl₃ (10 mL) over 20 h.The syringe needle was placed at the base of the condensorto ensureproper addition. After 20 h, the reaction was cooled to 50° C. and AcOH(0.046 mL, 0.812 mmol) was added. After 2 h, the reaction was cooled tort and washed with sat NaHCO₃ (30 mL), brine (30 mL) and dried overanhydrous Na₂SO₄. The lactone 15D was purified by flash chromatographyon silica gel eluting with hexanes/ethyl acetate (20:1→15:1) to give0.014 g(75%): IR (film): 2929, 1741, 1696, 1254, 1097 cm⁻¹; ¹HNMR(CDCl₃, 400 MHz) δ 6.95 (1H, s), 6.55 (1H, s), 5.48 (1H, m), 5.37(1H, m), 5.16 (1H, d, J=9.8 Hz), 14.17 (1H, d, J=8.3 Hz), 4.07 (1H, t,J=7.2 Hz), 3.02 (1H, t, J=7.2 Hz), 2.77 (1H, m), 2.70 (3H, s), 2.64 (2H,m), 2.29 (1H, m), 2.15 (1H, m), 2.12 (3H, s), 1.92 (1H, m), 1.71 (1H,m), 1.44 (2H, m), 1.26 (1H, m), 1.17 (3H, s), 1.12 (3H, s), 1.11 (3H, d,J=7.0 Hz), 0.91 (9H, s), 0.85 (9H, s), 0.09 (3H, s), 0.06 (6H, s), −0.04(3H, s); ¹³C NMR (CDCl₁, 100 MHz) δ 215.2, 171.9, 164.5, 152.5, 138.0,133.5, 123.8, 120.0, 116.7, 79.4, 76.2, 72.5, 53.5, 47.4, 39.9, 34.5,31.9, 31.5, 30.2, 27.7, 26.1, 25.9, 24.1, 23.8, 23.1, 22.6, 19.2, 18.5,18.2, 16.3, 14.9, 14.1, −3.7,−4.2, −4.7, −5.2.

EXAMPLE 55

[0351] Desmethyldesoxyepothilone A (16D):

[0352] To the lactone 15D (0.038 g, 0.056 mmol) in THF (2.0 mL) wasadded HF.pyridine (1.0 mL). After 2 h, the reaction was poured into satNaHCO₃ (30 mL) and extracted with CHCl₃ (5×20 mL). The organics weredried over Na₂SO₄. The crude diol 16D was purified by flashchromatography on silica gel eluting with hexaneslethyl acetate(3:1→2:1) to give 0.023 g (89%): IR (film): 3501, 2933, 1734, 1684,1290, 1248, 1045 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 6.95 (1H, s), 6.59 (1H,s), 5.40 (2H, m), 5.23 (1 H, dd, J=1.4, 9.5 Hz), 4.38 (1H, bd, J=11.1Hz), 3.78 (1H, t, J=6.9 Hz), 3.59 (1H, bs), 3.47 (1H, s), 2.99 (1H, q,J=7.0 Hz), 2.68 (3H, s), 2.66 (1H, m), 2.46 (1H, dd, J=11.4, 14.4 Hz),2.26 (1H, dd, J=2.2, 14.4 Hz), 2.22 (1H, m), 2.06 (3H, s), 1.96 (1H, m),1.49 (3H, m), 1.35 (3H, m), 1.30 (3H, s), 1.15 (3H, d, J=6.9 Hz), 1.06(3H, s); ¹³C NMR (CDCl₃₁ 100 MHz) δ 221.5, 170.3, 165.1, 151.8, 139.1,132.8, 125.2, 119.1, 115.5, 78.4, 72.5, 70.8, 53.8, 42.7, 39.6, 32.3,31.8, 28.3, 26.8, 24.8, 23.1, 19.0, 17.2, 16.0, 11.1.

EXAMPLE 56

[0353] Epoxide Formation:

[0354] Diol 16D (0.008 g, 0.017 mmol) was dissolved in CH₂Cl₂ (1.0 mL)and cooled to −60° C. Dimethyldioxirane (0.06 M, 0.570 mL, 0.0034 mmol)was then slowly added. The reaction temperature was slowly warmed to−25° C. After 2 h at −25° C., the volatiles were removed from thereaction at −25° C. under vacuum. The resulting residue was purified byflash chromatography on silica gel eluting with MeOH in CH₂Cl₂ (1%-2%)to give a 1.6:1 mixture of cis-epoxides 3D and the diastereomericcis-epoxide (0.0058 g, 74%). The diastereomeric epoxides were separatedby preparative-TLC eluting with hexanes/ethyl acetate (1:1) after 3elutions to give pure diastereomers:

[0355] Beta epoxide3D: IR (film): 3458, 2928, 1737, 1685, 1456, 1261,1150, 1043, 1014 cm⁻¹; ¹H NMR (CD₂Cl₂, 500 MHz) δ 7.01 (1H, s), 6.56(1H, s), 5.35 (1H, dd, J=2.3, 9.6 Hz), 4.30 (1H, dd, J=3.0, 5.7 Hz),3.85 (1H, m), 3.81 (1H, d, J=5.7 Hz), 3.42 (1H, d, J=2.0 Hz), 3.03 (1H,q, J=6.8 Hz), 2.97 (1H, m), 2.88 (1H, m), 2.67 (3H, s), 2.46 (1H, dd,J=9.0, 14.5 Hz), 2.33 (1H, dd, J=2.6, 14.5 Hz), 2.13 (1H, dt, J=3.0,15.0 Hz), 2.08 (3H, s), 1.82 (1H, m), 1.52 (6H, m), 1.41 (1H, m), 1.33(3H, s), 1.21 (4H, m), 1.12 (3H, d, J=7.0 Hz), 1.06 (3H, s); ¹³C NMR(CD₂Cl₂, 125 MHz) δ 221.9, 170.6, 165.6, 152.2, 138.3, 120.2, 116.6,77.3, 73.4, 69.9, 57.7, 55.3, 43.7, 39.7, 32.6, 32.0, 29.8, 27.2, 25.7,24.7, 22.5, 19.2, 19.0, 15.6, 15.6, 11.5;

[0356] Alpha epoxide: IR (film): 3439, 2918, 1735, 1684, 1455, 1262,1048, 1014 cm⁻¹; ¹H NMR (CD₂Cl₂, 500 MHz) δ 7.02 (1H, s), 6.56 (1H, s),5.62 (1H, d, J=8.1 Hz), 4.33 (1H, dd, J=2.7, 11.0 Hz), 3.85 (1H, t,J=5.9 Hz), 3.27 (1H, d, J=5.3 Hz), 3.11 (1H, m), 3.07 (1 H, d, J=7.0Hz), 3.04 (1H, s), 2.87 (1H, m), 2.68 (3H, s), 2.46 (1H, dd, J=11.1,14.1 Hz), 2.35 (1H, dd, J=2.3, 14.1 Hz), 2.11 (3H, s), 2.06 (1H, ddd,J=1.9, 4.5, 15.1 Hz), 1.87 (1H, m), 1.52 (6H, m), 1.38 (2H, m), 1.29(3H, s), 1.08 (3H, d, J=6.9 Hz), 1.03 (3 H, s); ¹³C NMR (CD₂Cl₂, 125MHz) δ 222.1, 170.2, 165.3, 152.5, 137.6, 119.7, 116.7, 76.7, 72.9,70.6, 57.1, 55.1, 44.7, 40.0, 32.1, 31.4, 30.0, 26.6, 25.5, 24.7, 21.3,19.3, 18.7, 15.7, 11.5.

EXAMPLE 57

[0357] Experimental Data for C-12 Hydroxy Epothilone Analogs

[0358] Propyl hydroxy compound 43:

[0359]¹H NMR (CDCl₃, 400 MHz) δ 6.96 (1H, s), 6.59 (1H, s), 5.16-5.23(2H, band), 4.28 (1H, m), 3.72 (1H, m), 3.63 (2H, t, J=6.3 Hz), 3.17(1H, dq, J=2.2, 0.5 Hz), 3.02 (1H, s), 2.70 (3H, s), 2.65 (2H, m), 2.46(1H, dd, J=10.9, 14.6 Hz), 2.29 (2H, m), 1.98-2.09 (6H, band), 1.60-1.91(6H, band), 1.35 (3H, s), 1.33 (3H, s), 1.18 (3H, d, J=6.8 Hz), 1.07(3H, s), 1.01 (3H, d, J=7.1 Hz); ¹³C NMR (CDCl₃, 100 MHz) δ 220.69,170.29, 165.00, 151.81, 141.63, 138.93, 120.64, 118.81, 115.52, 78.53,77.23, 73.93, 71.85, 62.26, 53.63, 41.57, 39.54, 37.98, 32.33, 32.14,31.54, 30.75, 29.67, 25.27, 22.89, 18.92, 17.67, 15.98, 15.74, 13.28; MSe/m 536.2, calc 535.29.

[0360] Hydroxy methyl compound 46:

[0361]¹H NMR (CDCl₃, 400 MHz) δ 6.97 (1H, s), 6.63 (1H, s), 5.43 (1H,dd, J=5.7, 9.1 Hz), 5.24 (1H, d, J=7.4 Hz), 4.31 (1H, d, J=9.7 Hz), 4.05(2H, dd, J=7.3, 31.0 Hz), 3.87 (1H, bs), 3.69 (1H, bs), 3.17 (1H, dd,J=2.0, 6.9 Hz), 3.03 (1H, s), 2.69 (3H, s), 2.63 (1H, m), 2.45 (1H, dd,J=11.2, 14.6 Hz), 2.37 (1H, m), 2.25 (2H, m), 2.11 (1H, m), 2.05 (3H,s), 1.78 (1H, m), 1.70 (1H, m), 1.35 (3H, s), 1.34 (2H, m), 1.29 (1H,m), 1.18 (3H, d, J=6.8 Hz), 1.06 (3H, s), 1.00 (3H, d, J=7.0 Hz); ¹³CNMR (CDCl₃, 100 MHz) δ 220.70, 170.16, 165.02, 151.63, 141.56, 138.41,121.33, 118.65, 115.33, 77.74, 77.25, 74.11, 71.37, 65.75, 53.86, 41.52,39.52, 37.98, 31.46, 27.70, 25.10, 22.86, 18.74, 17.20, 16.17, 15.63,13.41.

EXAMPLE 58

[0362] 4,4-Dimethyl-3,5-dioxoheptanoate, tert-butyl Ester 47.

[0363] t-Butyl 4-methyl-3-oxo-4-methyl pentanoate (22.5 g, 121 mmol) wasadded dropwise in 20 mL of dry THF to a slurry of NaH (6.29 g, 60%dispersion in mineral oil, 157.2 mmol) in 500 mL of dry THF. Thereaction mixture was stirred at 0° C. for 30 min and then the cold bathwas cooled to −50° C. Freshly distilled propionyl chloride (12.3 g,133.0 mmol) was added rapidly (neat) via syringe to the cold solution.The reaction was monitored by TLC and the cold bath was maintained below−30° C. until the reaction was complete. After 1 hr, the reaction wasquenched by pouring into a solution of saturated aqueous NH₄Cl andsubjected to an aqueous workup. Flash column chromatography with 2%EtOAc/hexanes afforded the desired tricarbonyl 42 (16.4 g, 67.8 mmol) in56% yield; ¹H NMR (400 MHz, CDCl₃): δ 12.43 (s, 0.20H), 5.07 (s, 0.20H),3.36 (s, 1.6H), 2.47 (q, J=7.13 Hz, 2H), 1.44 (s, 9H), 1.35 (s, 6H),1.03 (t, J=7.18 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 209.7, 202.9,166.1, 81.91, 62.53, 43.34, 31.67, 27.82 (3), 21.03 (2), 7.82; IR (neat)3411.8, 1742.6, 1718.5, 1702.0, 1644.2, 1460.6, 1156.1 cm⁻¹.

[0364] 4,4-Dimethyl-5-oxo-3-triethylsilyloxy-2-heptenoate, tert-butylEster 48.

[0365] The ester 47 (5.79 g, 23.9 mmol) in THF was added to a suspensionof NaH (60% in mineral oil, 1.24 g, 31.1 mmol) in THF (200 mL) at 0° C.After 20 min, the reaction was cooled to −50° C. and TESOTf (5.95 mL,26.32 mmol) was added. After an additional 20 min, the reaction waspoured into saturated aqueous NaHCO₃ (300 mL). This mixture wasextracted with Et₂O (2×200 mL) and the combined organic layers weredried over anhydrous MgSO₄O. The resulting oil was purified by flashcolumn chromatography on SiO2 eluting with Et₂O/hexanes (1:20 to 1:15)to give 6.65 g (78%) of the desired enol ether 48 as a clear oil; ¹H NMR(400 MHz, CDCl₃): δ 5.16 (s, 1H), 2.48 (q, J=7.2 Hz, 2H), 1.45 (s, 9H),1.24 (s, 6H), 1.02 (t, J=7.2 Hz, 3H), 0.95 (t, J=8.1 Hz, 9H), 0.74 (q,J=8.1 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃): δ 211.2, 169.4, 165.1, 97.77,78.93, 55.54, 30.33, 28.21, 22.94, 8.15, 6.78, 6.02; IR (neat) 1712,1619, 1384, 1243, 1150 cm⁻¹.

EXAMPLE 59

[0366](6R,7R,8S)-7-Hydroxy-5-oxo-4,4,6,8-tetramethyl-3-triethylsilyloxy-2,10-undecadienoate,tert-butyl ester 49.

[0367] The keto enol ether 48 (7.80 g, 22.0 mmol) in 175 mL of dry THFwas cooled to −30° C. in a cold bath (CO₂ (s)/CH₃CN) and then, a cooledsolution of LDA (27.2 mmol, 0.90 M in THF) was added dropwise viasyringe over 5 min. Immediately after the addition of the keto enolether, the reaction vessel was placed in a −120° C. cold bath(N2(liq)/pentane) and the reaction mixture was stirred for 10 min. Then,the aldehyde (2.0 g, 20.0 mmol) was added via syringe in 1 mL of dryTHF. The reaction was complete after 30 min and was quenched by pouringinto a solution of saturated aqueous NaHCO₃. The desired aldol product49 (6.0 g, 13.2 mmol) was isolated in 60% yield (yield of the majorproduct of a 5.5:1 mixture of diastereomers, epimeric at C-8) afterflash column chromatography with 6-8% Et₂O/hexanes; (major diastereomer,higher Rf); ¹H NMR (400 MHz, CDCl₃): δ 5.78 (m, 1H), 5.22 (m, 1H), 5.05(m, 2H), 3.37 (m, 2H), 3.18 (q, J=7.08 Hz, 1H), 2.52 (m, 1H), 1.85 (dt,J=14.0, 8.37 Hz, 1H), 1.62 (m, 1H), 1.55 (s, 3H), 1.46 (s, 9H), 1.22 (s,3H), 1.25 (s, 3H), 1.04 (d, J=6.90 Hz, 3H), 0.95 (t, J=7.94 Hz, 6H),0.76 (q, J=8.26 Hz, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 217.8, 168.1,164.6, 137.1, 116.2, 99.03, 79.21, 74.82, 56.74, 40.65, 37.39, 35.06,28.24, 22.53, 22.29, 14.77, 10.54, 6.95, 6.04; (minor diastereomer,lower Rf) ¹H NMR (400 MHz, CDCl₃): δ 5.73 (m, 1H), 5.23 (s, 1H), 5.02(m, 2H), 3.43 (d, J=8.74 Hz, 1H), 3.21 (m, 2H), 2.06 (m, 1H), 1.84 (m,1H), 1.62 (m, 1H), 1.55 (s, 3H), 1.46 (s, 9H), 1.27 (5, 3H), 1.24 (s,3H), 1.06 (d, J=6.91 Hz, 3H), 0.96 (t, J=8.06 Hz, 9H), 0.77 (q, J=7.53Hz, 6H); ¹³C NMR (100 MHz, CDCl₃): δ 217.8, 168.5, 165.0, 136.8, 116.8,99.46, 79.62, 75.47, 57.17, 41.43, 37.86, 35.58, 30.70, 28.66, 28.31,22.90, 22.74, 16.53, 11.77, 7.37, 6.44.

EXAMPLE 60

[0368] (6R,7R,8S)-7-Trichloroethoxyethylcarbonate-3,5-dioxo-4,4,6,8-tetramethyl-10-undEcenoate, tert-butyl Ester 50.

[0369] The alcohol 49 (1.61 g, 3.55 mmol) was dissolved in 20 mL of dryCH₂Cl₂ and cooled to 0° C. in an ice bath. Then, pyridine (1.12 g, 14.2mmol) and trichloroethoxyethylcarbonoyl chloride (TrocCl) (1.50 g, 7.10mmol) were added via syringe in that order. The reaction was stirred at0° C. for 5 min and then the ice bath was removed and the reaction wasallowed to come to rt and stir for 30 minutes. After this period oftime, TLC analysis showed the complete consumption of the startingmaterial. The reaction mixture was cooled to 0° C. and the TES enolether was hydrolyzed by the addition of 20 mL of 0.5 M methanolic HCl.The reaction mixture was stirred for 5 min at 0° C. and then quenched bypouring into a solution of saturated aqueous NaHCO₃. The desiredtricarbonyl 50 (1.54 g, 3.01 mmol) was isolated after an aqueous workupand flash column chromatography with 7-9% Et₂O/hexanes; ¹H NMR (400 MHz,CDCl₃): δ 12.63 (s, 0.25H), 5.70 (m, 1H), 5.15 (s, 0.25H), 5.08-4.88 (m,2H), 4.91 (dd, J=6.60, 5.01 Hz, 0.30H), 4.78 (m, 1H), 4.77 (dd, J=7.86,3.58 Hz, 0.70H), 4.72 (dd, J=11.8, 9.66 Hz, 1H), 3.48 (d, J=16.2 Hz,0.75H), 3.42 (d, J=16.2 Hz, 0.75H), 3.36 (m, 0.30H), 3.30 (m, 0.70H),1.88 (m, 2H), 1.50 (s, 3H), 1.46 (s, 9H), 1.39 (s, 3H), 1.12 (d, J=6.88Hz, 0.70H), 1.10 (d, J=6.88 Hz, 1.3H), 0.93 (d, J=6.63 Hz, 1.3H), 0.88(d, J=6.86 Hz, 0.70H); ¹³C NMR (100 MHz, CDCl₃): δ 210.5, 209.5, 203.16,178.3, 172.6, 166.2, 154.1, 135.9, 135.6, 117.2, 116.9, 94.69, 94.56,90.69, 82.68, 81.98, 81.65, 81.53, 63.58, 54.34, 46.56, 41.99, 41.62,36.41, 35.84, 34.49, 34.44, 31.56, 28.23 (3), 27.94 (3), 22.62, 22.08,21.56, 20.80, 15.95, 15.58, 14.09, 13.02, 12.98, 11.35; IR (neat)1757.9, 1718.9, 1700.2, 1642.2, 1620.7, 1250.6, 1156.3 cm⁻¹.

EXAMPLE 61

[0370] TBS Vinyl Iodide 51.

[0371] n-BuLi (1.6 M in hexanes, 7.69 mL, 12.3 mmol) was added to asuspension of ethyl triphenylphosphonium iodide (5.15 g, 12.3 mmol) inTHF (50 mL) at 25° C. After 20 min, the clear red solution wastransferred dropwise via syringe to a vigorously stirred solution of 12(3.12 g, 12.3 mmol) in THF (100 mL) at-78° C. The resulting pale yellowsuspension was stirred rapidly and warmed to 20° C. Then, NaHMDS (1.0 Msoln in THF, 12.3 mL, 12.3 mmol) was added dropwise via syringe. Duringthe addition of the NaHMDS, the reaction mixture changed fromayellow-orange slurry and to bright red solution. The TBS aldehyde (D.-S. Su et al., Angew. Chem. Int. Ed. Engl., 1997, 36, 757; 2.00 g, 6.15mmol) was then added in THF. After 30 min, the reaction mixture waspoured into hexanes (100 mL) and H₂O (0.5 mL) was added. The solutionwas then passed through a plug of SiO2 eluting with 2:1 hexanes/Et₂O.The iodide was purified by flash chromatography on SiO2 eluting withhexanes/ethyl acetate (20:1 to 15:1) to give the vinyl iodide 51 (1.46g, 55%) as a yellow oil:

[0372]¹H NMR (400 MHz, CDCl₃): δ 6.95 (s, 1H), 6.50 (s, 1H), 5.45 (dt,J=1.5, 6.8 Hz, 1H), 4.22 (t, J=6.4 Hz, 1H), 2.73 (s, 3H), 2.48 (s, 3H),2.39 (m, 2H), 2.02 (s, 3H), 0.90 (s, 9H), 0.06 (3, s), 0.02 (3, s); ¹³CNMR (100 MHz, CDCl₃): δ 164.86, 153.46, 142.17, 132.54, 119.23, 115.68,102.79, 77.70, 44.40, 39.09, 26.35, 19.65, 18.63, 14.54, −4.59, −4.84;IR (neat) 2928, 1470, 1252, 1068 cm⁻¹.

[0373] Post-Suzuki, C-15 Hydroxy Tricarbonyl 52.

[0374] 9-BBN (0.5 M soln in THF, 6.68 mL, 3.34 mmol) was added over a 45min period to a solution of the olefin 50 (1.43 g, 2.78 mmol) in THF (15mL) at 25° C. After 2 h, TLC analysis revealed the complete consumptionof the starting olefin. In a separate flask, containing the vinyl iodide51 (1.20 g, 2.80 mmol) and DMF (20 mL), were added successively and withvigorous stirring: Cs₂CO₃ (1.82 g, 5.60 mmol), Pd(dppf)₂Cl₂ (0.454 g,0.56 mmol), AsPh₃ (0.171 g, 0.56 mmol) and H₂O (1.82 mL, 0.1 mmol).

[0375] Then the borane solution, prepared above, was added rapidly tothe vigorously stirred solution containing the vinyl iodide. After 2 h,the reaction was complete and the reaction mixture was poured into Et₂O(300 mL) and washed with H₂O (3×200 mL), brine (1×50 mL) and dried overanhydrous MgSO₄. This crude product was purified by flash columnchromatography on SiO₂ eluting with hexanes/ethyl acetate (18:1 to 13:1to 10:1) to afford the TBS protected coupled product as an impuremixture which was taken on to the next step.

[0376] The crude TBS protected coupled product (˜2.78 mmol) wasdissolved in 0.36 N HCl in MeOH (30 mL) at 25° C. After 3.5 h, themixture was poured into a solution of saturated aqueous NaHCO₃ andextracted with CHCl₃ (4×60 mL). The combined organic layers were washedonce with brine (50 mL) and dried over anhydrous Na₂SO₄. The diol waspurified by flash column chromatography on SiO₂ eluting withhexaneslethyl acetate (4:1 to 3:1 to 2:1) to give the pure product 52 asa clear oil (0.910 g, 46% for two steps): ¹H NMR (400 MHz, CDCl₃): δ6.96 (s, 1H), 6.56 (s, 1H), 5.16 (t, J=6.9 Hz, 1H), 4.83 (d, J=11.9 Hz,1H), 4.75 (dd, J=3.4, 8.0 Hz, 1H), 4.70 (d, J=11.9 Hz, 1H), 4.14 (t,J=6.4 Hz, 1H), 3.45 (q, J=13.2 Hz, 2H), 3.32 (m, 1H), 2.72 (s, 3H), 2.32(t, J=6.5 Hz, 2H), 2.04 (s, 3H), 2.01 (m, 2H), 1.74 (m, 2H), 1.69 (s,3H), 1.45 (s, 9H), 1.38 (s, 6H), 1.09 (d, J=6.9 Hz, 3H), 0.93 (d, J=6.9Hz, 3H); ¹³CNMR (100 MHz, CDCl₃): δ 209.51, 203.04, 166.15, 164.39,154.14, 152.72, 141.71, 138.24, 120.70, 118.76, 115.28, 94.54, 81.85,77.31, 76.57, 63.41, 54.16, 46.47, 41.48, 34.56, 33.95, 31.98, 31.53,27.85, 24.85, 23.45, 21.47, 20.75, 19.04, 15.60, 14.33, 11.35; IR (neat)3546, 3395, 1756, 1717, 1699, 1644, 1621, 1506, 1456, 1251 cm⁻¹.

EXAMPLE 62

[0377] Noyori C-3/C-15 Diol Product 53.

[0378] The diketone 52 (0.900 g, 1.27 mmol) was dissolved in 0.12 N HClin MeOH (10 mL) at 25° C. The RuBINAP catalyst (0.018 M in THF, 1.0 mL,0.018 mmol) was then added and the mixture transferred to a Parrapparatus. The vessel was purged with H₂ for 5 min and then pressurizedto 1200 psi. After 12 h at 25° C., the reaction was returned toatmospheric pressure and poured into a saturated solution of NaHCO₃.This mixture was extracted with CHCl₃ (4×50 mL) and the combined organiclayers were dried over anhydrous Na₂SO₄. The product was purified byflash column chromatography on silica gel eluting with hexanes/ethylacetate (4:1 to 2:1) to give 0.75 g (81%) of the hydroxy ester 53 as awhite foam; ¹H NMR (400 MHz, CDCl₃): δ 6.94 (s, 1H), 6.55 (s, 1H), 5.15(t, J=6.9 Hz, 1H), 4.85 (t, J=5.3 Hz, 1H), 4.81 (d, J=12.0 Hz, 1H), 4.71(d, J=12.0 Hz, 1H), 4.12 (m, 2H), 3.43 (m, 2H), 2.70 (s, 3H), 2.37 (dd,J=2.2, 6.2 Hz, 1H), 2.30 (t, J=6.7 Hz, 2H), 2.24 (dd, J=10.6, 16.2 Hz,1H), 2.03 (s, 3H), 1.99 (m, 2H), 1.68 (s, 3H), 1.44 (s, 9H), 1.18 (s,3H), 1.16 (s, 3H), 1.09 (d, J=6.8 Hz, 3H), 0.94 (d, J=6.8 Hz, 3H); ¹³CNMR (100 MHz, CDCl₃): δ 5215.95, 172.39, 164.39, 154.21, 152.74, 141.70,138.33, 120.59, 118.77, 115.27, 94.64, 82.98, 81.26, 76.51, 72.78,51.82, 41.40, 37.36, 34.66, 33.96, 32.08, 31.10, 30.20, 27.96, 25.06,23.45, 21.73, 21.07, 19.17, 19.01, 16.12, 15.16, 14.33, 12.17;IR(neat)3434.0, 1757.5, 1704.5, 1249.9, 1152.8 cm⁻¹.

EXAMPLE 63

[0379] C-3/C-15 Bis(TES) Carboxylic Acid 54.

[0380] 2,6-Lutidine (0.48 g, 4.5 mmol) and TESOTf (0.59 g, 2.25 mmol)were added successively to a cooled solution of the diol 53 (164 mg,0.225 mmol) in CH₂Cl₂ (2.5 mL) at −78° C. The reaction mixture wasstirred at −78° C. for 5 min and then warmed to rt. The reaction wasstirred at rt for 6 hr and then quenched with saturated aqueous NH₄Cland subjected to an aqueous workup. The crude product was concentratedin vacuo and subjected directly to the next set of reaction conditions;¹H NMR (400 MHz, CDCl₃): δ 6.96 (s, 1H), 6.66 (s, 1H), 5.04 (t, J=6.93Hz, 1H), 4.90 (d, J=12.0 Hz, 1H), 4.77 (dd, J=7.99, 3.21 Hz, 1H), 4.66(d, J=12.0 Hz, 1H), 4.46 (m, 1H), 4.10 (dq, J=12.3, 7.11 Hz, 2H), 3.42(m, 1H), 2.70 (s, 3H), 2.60 (dd, J=16.7, 2.34 Hz, 1H), 2.34 (dd, J=16.7,7.94 Hz, 1H), 2.27 (dd, J=14.0, 6.97 Hz, 1H), 2.18 (m, 1H), 2.09 (m,1H), 2.04 (s, 1H), 1.95 (s, 3H), 1.82 (m, 2H), 1.61 (s, 3H), 1.44 (m,2H), 1.27-1.22 (m, 4H), 1.14 (d, J32 8.45 Hz, 3H), 1.11 (d, J=6.81 Hz,2H), 1.04 (d, J=6.88 Hz, 2H), 1.15-1.01 (m, 2H), 0.94 (t, J=7.92 Hz,18H), 0.65-0.57 (m, 12H); ¹³C NMR (100 MHz, CDCl₃): δ 215.11, 175.34,165.00, 154.14, 152.80, 142.60, 136.84, 121.31, 118.79, 114.60, 94.77,81.60, 79.06, 76.64, 73.87, 54.19, 41.18, 39.56, 35.09, 34.52, 32.29,31.95, 24.76, 23.62, 22.55, 18.95, 18.64, 15.87, 13.69, 11.33, 6.94,6.83, 5.07, 4.76; IR (neat) 3100-2390, 1756.8, 1708.8, 1459.3, 1250.6,816.1 cm⁻¹.

EXAMPLE 64

[0381] C-15 Hydroxy Acid for Macrolactonization 55.

[0382] The crude bis(triethylsilyl)ether 54, prepared above, wasdissolved in 5 mL of dry THF and then cooled to ⁰° C. Then, 1 mL of 0.12M HCl/MeOH was added. The reaction mixture was stirred at 0° C. for 20min and then checked for completion. TLC analysis at this time revealedthe complete consumption of starting material. The reaction was quenchedby pouring into a solution of saturated aqueous NaHCO₃ and subjected toan aqueous workup. Flash column chromatography with 25 to 30:1CHCl₃/MeOH afforded the desired carboxylic acid 55 in 77% yield; ¹H NMR(400 MHz, CDCl₃): δ 6.96 (s, 1H), 6.69 (1, s), 5.11 (t, J=6.9 Hz, 1H),4.91 (d, J=12.0 Hz, 1H), 4.71 (dd, J=3.1, 8.2 Hz, 1H), 4.64 (d, J=12.0Hz, 1H), 4.42 (d, J=5.9 Hz, 1H), 4.10 (m, 1H), 3.43 (m, 1H), 2.71 (s,3H), 2.57 (dd, J=2.1, 10.5 Hz, 1H), 2.25 (m, 3H), 2.11 (m, 1H), 1.98 (s,3H), 1.95 (m, 2H), 1.72 (m 1), 1.67 (s, 3H), 1.45 (m, 2H), 1.16 (s, 3H),1.13 (s, 3H), 1.09 (d, J=6.7 Hz, 3H), 0.99 (d, J=6.7 Hz, 3H), 0.95 (t,J=7.9 Hz, 9H), 0.64 (dq, J=2.3, 7.9 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃): δ215.11, 176.00 (165.10, 154.18, 152.35, 142.24, 138.55, 120.74, 118.21,115.02, 94.76, 81.91, 76.86, 76.63, 73.95, 54.08, 41.28, 39.64, 34.73,34.16, 32.02, 31.67, 24.71, 23.41, 22.49, 19.17, 18.62, 15.71, 14.86,11.20, 6.93, 5.05); IR (neat) 3400-2390, 1755.9, 1703.8, 1250.4, 735.4cm⁻¹.

EXAMPLE 65

[0383] C-3 Triethylsilyl/C-7 TrichloroethoxyethylcarbonateMacrolactonization Product 56.

[0384] Triethylamine (155 mg, 1.53 mmol) and 2,4,6-trichlorobenzoylchloride (312 mg, 1.28 mmol) were added to a solution of the hydroxyacid 55 (198 mg, 0.256 mmol) in 3.6 mL of dry THF. The reaction mixturewas stirred for 0.25 h at rt and then diluted with 45 mL of dry toluene.The resultant solution was added slowly dropwise, via syringe pump, over3 hr to a stirred solution of DMAP (328 mg, 2.68 mmol) in 145 mL of drytoluene. After the addition of the substrate was complete, the reactionwas stirred for an additional 0.5 h and then taken up in an equal volumeof Et₂O and washed with 1N HCl (1×), saturated aqueous NaHCO₃ (1×), andbrine (1×). The organic layer was dried over MgSO₄ and concentrated invacuo. Flash column chromatography of the crude product with 10%EtOAc/hexanes afforded the desired macrolactone (153 mg, 0.20 mmol) in78% yield; ¹H NMR (400 MHz, CDCl₃): δ 6.96 (s, 1H), 6.53 (s, 1H), 5.20(m, 2H), 5.04 (d, J=10.2 Hz, 1H), 4.84 (d, J=12.0 Hz, 1H), 4.78 (d,J=12.0 Hz, 1H), 4.07 (m, 1H), 3.32 (m, 1H), 2.86-2.63 (m, 3H), 2.70 (s,3H), 2.48 (m, 1H), 2.11 (s, 3H), 2.04 (dd, J=6.17, 14.7 Hz, 1H), 1.73(m, 4H), 1.66 (s, 3H), 1.25 (m, 2H), 1.19 (s, 3H), 1.15 (s, 3H), 1.12(d, J=6.68 Hz, 3H), 1.01 (d, J=6.83 Hz, 3H), 0.89 (t, J=8.00 Hz, 9H),0.58 (q, J=7.83 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃): δ 212.75, 170.66,164.62, 154.60, 152.52, 140.29, 138.44, 119.81, 119.38, 116.28, 94.84,86.44, 80.14, 76.59, 76.10, 53.55, 45.89, 39.23, 35.47, 32.39, 31.69,31.57, 31.16, 29.68, 27.41, 25.00, 23.44, 22.94, 19.23, 18.66, 16.28,14.83, 6.89, 5.22; IR (neat) 1760.5, 1742.6, 1698.0, 1378.8, 1246.2,1106.0, 729.8 cm⁻¹.

EXAMPLE 66

[0385] SmI₂ Mediated Deprotection of Troc Group 57.

[0386] Samarium metal (0.334 g, 2.22 mmol) and iodine (0.51 g, 2.0 mmol)in 25 mL of dry, deoxygenated THF were stirred together vigorously for2.5 hr at ambient temperature. During this period of time, the reactionmixture progressed from a dark orange to an olive green to deep bluecolor. The resultant deep blue solution of SmI2 was used directly in thefollowing reaction. 5 ml2 (25 mL of a 0.08 M stock solution, 2.0 mmol)was added rapidly via syringe to a stirred solution of the macrolactone57 (200 mg, 0.26 mmol) and a catalytic amount of NiI₂ (10 mg) in 10 mLof dry THF at −78° C.

[0387] The resultant deep blue solution was maintained at −78° C. withcontinued vigorous stirring for 2.5 hr. TLC analysis at this timerevealed the complete consumption of the starting material and formationof a single, lower Rf product. The reaction mixture was quenched withsaturated aqueous NaHCO₃ and subjected to an aqueous workup. Flashcolumn chromatography with 25% EtOAc/hexanes afforded the desiredalcohol 57 (143 mg, 0.24 mmol) in 91% yield; ¹H NMR (400 MHz, CDCl₃): δ6.95 (s, 1H), 6.54 (s, 1H), 5.15 (m, 1H), 5.05 (d, J=10.15 Hz, 1H), 4.08(dd, J=10.1, 2.66 Hz, 1H), 3.87 (m, 1H), 3.01 (s, 1H), 3.06 (m, 1H),2.83-2.65 (m, 3H), 2.70 (s, 3H), 2.44 (m, 1H), 2.10 (s, 3H), 2.07 (m,1H), 1.83 (m, 1H), 1.77 (m, 1H), 1.71 (m, 1H), 1.64 (s, 3H), 1.60 (s,1H), 1.37 (m, 1H), 1.31 (m, 1H), 1.20 (m, 1H), 1.15 (s, 3H), 1.14 (m,5H), 1.02 (d, J=7.02 Hz, 3H), 0.89 (t, J=7.97 Hz, 9H), 0.64-0.52 (m,6H); ¹³C NMR (100 MHz, CDCl₃): δ 218.34, 170.73, 164.59, 152.46, 139.07,138.49, 120.48, 119.54, 116.00, 79.31, 75.81, 73.48, 53.62, 42.98,39.48, 39.01, 32.85, 32.41, 31.20, 26.12, 24.26, 22.01, 22.46, 19.18,16.44, 15.30, 13.99, 6.98 (3), 5.27 (3); IR (neat) 3524.0, 1740.3,1693.4, 1457.2, 1378.4, 733.2 cm⁻¹.

EXAMPLE 67

[0388] Desoxyepothilone B 2C.

[0389] The TES protected alcohol 57 (143 mg, 0.24 mmol) was dissolved in2 mL of dry THF in a plastic reaction vessel and cooled to 0° C. in anice bath. The resultant solution was treated with 1 mL of HF-pyridine.The reaction mixture was stirred for 80 min at 0° C. and then quenchedby pouring into a saturated aqueous solution of NaHCO₃. An aqueousworkup followed by flash column chromatography with 10% EtOAc/hexanesafforded desoxyepothilone B (112 mg, 0.23 mmol) in 95% yield. Theresultant product exhibited a ¹H NMR spectrum identical to that ofauthentic desoxyepothilone B.

[0390] Total Synthesis of Desoxyepothilone B

EXAMPLE 68

[0391] tert-Butyl 4-methyl-3-oxopentanoate (1E).

[0392] Meldrum's acid (80 g, 555 mmol) was dissolved in 600 mL of CH₂Cl₂and cooled to ⁰° C. Freshly distilled pyridine (87.8 g, 1.11 mol) wasadded to the CH₂Cl₂ solution and then iso-butyryl chloride (65.0 g,610.5 mmol) was added to the mixture via a pressure equalizing additionfunnel. The reaction was stirred at 0° C. for 1 hr and then warmed to rtand stirred for 2 hr. Then, the reaction was quenched with water (200mL) and washed with 0.5 M HCl (×2), water (×1), and brine (×1). Theorganize layer was dried over MgSO₄ and concentrated in vacuo. The crudeproduct was azeotropically dried with benzene (250 mL), and thendissolved in 200 mL of benzene and 200 mL of tert-butanol was added. Theresultant reaction was heated at reflux for 4 hr. After this period, thevolatiles were removed in vacuo and the product then distilled on thehigh vacuum pump (bp 62-63° C., 0.1 mm Hg). The desired β-keto ester 1Ewas obtained (58.6 g, 315.5 mmol) in 57% yield as a clear, colorless,light oil.

EXAMPLE 69

[0393] tert-Butyi 4,4-dimethy-3,5-dioxoheptanoate (2E).

[0394] β-Keto ester 1E (55.0 g, 295.3 mmol) was added dropwise in 50 mLof dry THF to a slurry of NaH (9.7 g, 60% dispersion in mineral oil,383.9 mmol) in 1.15 L of dry THF. The reaction mixture was stirred at 0°C. for 30 min and then the cold bath was cooled to −50° C. Propionylchloride (27.3 g, 295.3 mmol) was added rapidly (neat) by syringe to thecold solution. The reaction was monitored by TLC and the cold bath wasmaintained below −30° C. until the reaction was complete. After 1 hr,the reaction was quenched by pouring into a solution of saturatedaqueous NH₄Cl and subjected to an aqueous workup. The aqueous layer wasextracted with Et₂O (x2, 200 mL).

[0395] Flash column chromatography with 2% EtOAc/hexanes afforded thedesired tricarbonyl 2E (50.7 g, 209.6 mmol) in 71% yield; ¹H NMR (400MHz, CDCl₃); δ 12.43 (s, 0.20H), 5.07 (s, 0.20H), 3.36 (s, 1.6H), 2.47(q, J=7.13 Hz, 2H), 1.44 (s, 9H), 1.35 (s, 6H), 1.03 (t, J=7.18 Hz, 3H);¹³C NMR (100 MHz, CDCl₃): δ 209.7, 202.9, 166.1, 81.91, 62.53, 43.34,31.67, 27.82 (3), 21.03 (2), 7.82; IR (neat) 3411.8, 1742.6, 1718.5,1702.0, 1644.2, 1460.6, 1156.1 cm⁻¹

EXAMPLE 70

[0396] tert-Butyl-4,4-dimethyl-3-methoxy-5-oxo-2-heptenoate (3E).

[0397] Trimethylsilyldiazomethane (TMSCHN₂, 46.2 mL of a 2.0 M solutionin THF, 92.4 mmol) was added by syringe to a stirred solution of thetricarbonyl (16.0 g, 66.0 mmol) and diisopropylethylamine (Hunig's base,16.1 mL, 92.4 mmol) in 330 mL of a 9:1 solution of acetonitrile:methanolat rt. The resultant reaction mixture was stirred at rt for 18-20 hr.The reaction mixture was then quenched with saturated aqueous NaHCO₃ andthe enol ether extracted with Et₂O (×3, 50 mL). The combined organiclayers were washed with brine and then dried of MgSO₄, filtered andconcentrated in vacuo. Flash column chromatography of the crude product(2% EtOAc/hexanes) afforded the desired enol ether 3E (12.5 g, 48.4mmol) in 74% yield; ¹H NMR (400 MHz, CDCl₃): δ 5.18 (s, 1H), 3.88 (s,3H), 2.45 (q, J=7.33 Hz, 2H), 1.48 (s, 9H), 1.25 (s, 6H), 1.02 (t,J=7.21 Hz, 3H).

EXAMPLE 71

[0398](67R,7R,8S)-7-Hydroxy-5-oxo-4,4,6,8-tetramethy-3-triethysilyloxy-2,10-undecadienoate,tert-butyl ester (6E).

[0399] The keto enol ether 3E (8.0 g, 31.3 mmol) in 750 mL of dry THFwas cooled to −30° C. in a cold bath (CO₂ (s)/CH₃CN) and then a solutionof LDA (37.5 mmol, 0.90 M in THF) was added dropwise via syringe over 10min. The reaction mixture was stirred at −30 to −33° C. for 20 min. Thenthe reaction vessel was placed in a −120° C. cold bath (N₂(liq)/pentane)and the reaction mixture was stirred for 10 min. Finally the aldehyde 5(3.6 g, 36.7 mmol; aldehyde 5E was readily prepared according to theprocedure outlined in: Lin, N. -H., et al., J. Am. Chem. Soc. 1996, 118,9062.) was added via syringe in 5 mL of CH₂Cl₂. The reaction wascomplete after 10 min and was quenched by pouring into a solution ofsaturated aqueous NH₄Cl. The desired aldol product 6E (5.2 g, 14.7 mmol)was isolated in 47% yield (yield of the major product of a 5.5:1 mixtureof diastereomers, epimeric at C-8) after flash column chromatographywith 6-5% EtOAc/hexanes; (major diasteromer, high Rf); ¹H NMR (400 MHz,CDCl₃): δ 5.78 (m, 1H), 5.18 (s, 1H), 4.98 (m, 2H), 3.90 (s, 3H), 3.37(m, 1H), 3.35 (s, 1H), 3.35 (s, 1H), 3.12 (q, J=7.74 Hz, 1H), 2.53 (m,1H), 1.87 (dt, J=13.8, 8.47 Hz, 1H), 1.61 (m, 1H), 1.55 (s, 1H), 1.48(s, 9H), 1.28 (s, 3H), 1.27 (s, 3H), 1.05 (d, J=6.91 Hz, 3H), 0.079 (d,J=6.76 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 217.7, 171.2, 164.8, 137.0,116.3, 97.29, 80.22, 74.67, 62.17, 56.05, 41.05, 37.31, 34.99, 28.13,22.69, 22.67, 15.00, 10.44; (minor diastereomer, low Rf): ¹H NMR (400MHz, CDCl₃): δ 5.76 (m, 1H), 5.19 (s, 1H), 5.06 (m, 2H), 1.48 (s, 3H),3.41 (m, 1H), 3.17 (m, 1H), 3.12 (m, 1H), 2.11 (m, 1H), 1.86 (m, 1H),1.63 (m, 1H), 1.48 (s, 9H), 1.28 (s, 3H), 1.27 (s, 3H), 1.07 (s, J=6.91Hz, 3H), 0.99 (d, J=6.64 Hz, 3H).

EXAMPLE 72

[0400](6R,7R,8S)-7-(2,2,2-Trichloroethoxycarbonate)-5-oxo-4,4,6,8-tetramethyl-3-triethylsi-lyloxy-2,10-undecadienoate,tert-butyl ester.

[0401] Alcohol 6E (5.2 g, 14.7 mmol) was dissolved in 70 mL of dryCH₂Cl₂ and cooled to 0° C. in an ice bath. Then, pyridine (4.65 g, 58.8mmol) and trichloroethoxyethylcarbonoyl chloride (TrocCl) (6.23 g, 29.4mmol) were added by syringe in that order. The reaction was stirred at0° C. for 5 min and then the ice bath was removed and the reaction wasallowed to come to rt and stir for 30 minutes. After this period oftime, TLC analysis showed the complete consumption of the startingmaterial. The reaction mixture was quenched by pouring it into asolution of saturated aqueous NaHCO₃. Flash column chromatography with3% EtOAc/hexanes through a short plug of silica gel afforded the desiredenol ether which was subjected immediately to hydrolysis; ¹H NMR (400MHz, CDCl₃): δ 5.71 (m, 1H), 5.21 (s, 1H), 5.02 (m, 2H), 4.86 (d, J=11.9Hz, 1H), 4.85 (m, 1H), 4.72 (d, J=11.9 Hz, 1H), 3.91 (s, 3H), 3.25 (m,1H), 2.26 (m, 1H), 1.87 (m, 1H), 1.81 (m, 1H), 1.48 (s, 9H), 1.31 (s,3H), 1.26 (2, 3H), 1.11 (d, J=6.85 Hz, 3H), 0.91 (d, J=6.64 Hz, 3H).

EXAMPLE 73

[0402](6R,7R,8S)-7-Trichloroethoxyethylcarbonate-3,5-dioxo-4,4,6,8-tetramethyl-10-undecenoate,tert-butyl ester (7E).

[0403] The Troc-protected enol ether (as above) was dissolved in acetoneand treated with 300 mg (catalytic) of p-TsOH at rt for 5-6 hrs. Thereaction was monitored by TLC and after complete consumption of thestarting enol ether was apparent, the reaction mixture was quenched withsaturated aqueous NaHCO₃. The desired tricarbonyl 7E (6.8 g, 12.8 mmol),87% (2 steps) was isolated after an aqueous workup and flash columnchromatography with 7-9% EtOAc/hexanes; ¹H NMR (400 MHz, CDCl₃): δ 12.63(s, 0.25H), 5.70 (m, 1H), 5.15 (s, 0.25H), 5.08-4.88 (m, 2H), 4.91 (dd,J=6.60, 5.01 Hz, 0.30H), 4.78 (m, 1H), 4.77 (dd, J 7.86, 3.58 Hz,0.70H), 4.72 (dd, J=11.8, 9.66 Hz, 1H), 3.48 (d, J=16.2 Hz, 0.75H), 3.42(d, J=16.2 Hz, 0.75H), 3.36 (m, 0.30H), 3.30 (m, 0.70H), 1.88 (m, 2H),1.50 (s, 3H), 1.46 (s, 9H), 1.39 (s, 3H), 1.12 (d, J=6.88 Hz, 0.70H),1.10 (d, J=6.88 Hz, 1.3H), 0.93 (d, J=6.63 Hz, 1.3H), 0.88 (d, J=6.86Hz, 0.70H); ¹³C NMR (100 MHz, CDCl₃): δ 210.5, 209.5, 203.16, 178.3,172.6, 166.2, 154.1, 135.9, 135.6, 117.2, 116.9, 94.69, 94.56, 90.69,82.68, 81.98, 81.65, 81.53, 63.58, 54.34, 46.56, 41.99, 41.62, 36.41,35.84, 34.49, 34.44, 31.56, 28.23 (3), 27.94 (3), 22.62, 22.08, 21.56,20.80, 15.95, 15.58, 14.09, 13.02, 12.98, 11.35; IR (neat) 1757.98,1718.9, 1700.2, 1642.2, 1620.7, 1250.6, 1156.3 cm⁻¹

EXAMPLE 74

[0404] Allylic Alcohol (16E).

[0405] A mixture of (S)-(−)-1,1′-bi-2-naphthol (1.37 g, 4.8 mmol),Ti(O-1-Pr)₄ (1.36 g, 4.8 mmol), and 4 A sieves (11 g) in CH₂Cl₂ (300 mL)was heated at reflux for 1 h. The mixture was cooled to rt and aldehyde15E (8.0 g, 47.9 mmol; prepared according to the procedure outlined inan earlier Danishefsky synthesis of the epothilones: Meng, D.;Sorensen., E. J.; Bertinato, P.; Danishefsky, S. J. J. Org. Chem. 1996,61, 7998) was added. After 10 min, the suspension was cooled to −78° C.,and allyl tri-n-butyltin (20.9 g, 67.1 mmol) was added. The reactionmixture was stirred for 10 min at −78° C. and then placed in a 20° C.freezer for 70 h. Saturated aqueous NaHCOc solution (2 mL) was added,and the mixture was stirred for 1 h, poured over Na₂SO₄, and thenfiltered through a pad of MgSO₄ and celite. The crude material waspurified by flash chromatography with EtOAc/hexanes(5%→10%→15%→20%→25%→30%, two column volumes each) to give alcohol 16E asa yellow oil (6.0 g, 88.0 mmol) in 60% yield; [α]_(D)=−15.9 (c. 4.9,CHCl₃); IR (film) 3360, 1641, 1509, 1434, 1188, 1017, 914 cm⁻¹; ¹H NMR(500 MHz, CDCl₃, 25° C.) δ 6.92 (s, 1H), 6.55 (s, 1H), 5.82 (m, 1H),5.13 (dd, J=17.1, 1.3 Hz, 1H), 5.09 (d, J=10.2 Hz, 1H), 4.21 (t, J=6.0Hz, 1H), 2.76 (br s, 1H), 2.69 (s, 3H), 2.40 (m, 2H), 2.02 (s, 3H); ¹³CNMR (125 MHz, CDCl₃, 25° C.) δ 164.5, 152.6, 141.5, 134.6, 119.2, 117.6,115.3, 76.4, 39.9, 19.0, 14.2; HRMS calcd. for C₁₁H₁₅NOS: 209.0874found: 209.0872 (M+H).

EXAMPLE 75

[0406] TBS Allylic Ether (17E).

[0407] Alcohol 16E (5.70 g, 27.3 mmol) was dissolved in 50 mL of dryCH₂Cl₂ and cooled to −78° C. Then, 2,6-lutidine (7.6 g, 70.9 mmol) andTBSOTf (9.36 g, 35.4 mmol) were added via syringe successively and inthat order. The reaction mixture was stirred at this temperature for 30minutes and then quenched by pouring the reaction mixture into saturatedaqueous NaHCO₃. An aqueous workup followed by flash columnchromatography with 2% EtOAc/hexanes afforded the desired TBS ether(7.37 g, 22.8 mmol) in 84% yield. The allylic alcohol, 17E, could alsobe prepared according to the general procedure outlined in the followingreferences: (a) Racherla, U. S.; Brown, H. C. J. Org. Chem. 1991, 56,401. (b) Yang, Z.; He, Y.; Vourloumis, D.; Vallberg, H.; Nicolaou, K. C.Angew. Chem., Int. Ed. Engl., 1997, 36, 166.

EXAMPLE 76

[0408] Aldehyde (18E).

[0409] To a solution of TBS ether 17E (7.37 g, 22.8 mmol) in acetone(150 mL) at 0° C. was added 6.7 g of a 60% solution ofN-methyl-morpholine-N-oxide (NMO) in water (34.2 mmol), OsO4 (0.039 M inTHF, 6 mL, 0.23 mmol). The resultant mixture was stirred at 0° C. for 2h and then quenched with saturated aqueous Na₂SO₃ solution (100 mL). Thesolution was poured into H₂O (100 mL) and extracted with EtOAc (8×50mL). The combined organic layer was dried over MgSO₄, filtered,concentrated, and flashed through a short plug of silica gel to afford(7.8 g, 21.8 mmol) the crude diol in 96% yield.

[0410] To a solution of the crude diol (7.8 g, 21.8 mmol; the oxidationprocedure described here may also be acomplished with NaIO₄ as outlinedin a previous Danishefsky synthesis of the epothilones: Meng, D., etal., J. Ann. Chem. Soc. 1997,119, 10073.) in 400 mL benzene at 0° C. wasadded Pb(OAc)₄ (19.4 g, 43.7 mmol) and Na₂CO₃ (9.24 g, 87.2 mmol). Thereaction mixture was stirred at 0° C. for 10 min and then rt for 1.5 hr.After this period of time, the reaction mixture was quenched by pouringinto brine. The reaction was filtered through Celite™ and then theresultant aqueous layer was extracted with EtOAc (5×50 mL) dried overMgSO₄. Flash column chromatography on silica gel with 20% EtOAc/hexaneson a short pad of silica gave the aldehyde 18E as a yellow oil (5.02 g,15.5 mmol) in 71% yield.

EXAMPLE 77

[0411] TBS vinyl iodide (19E).

[0412] n-BuLi (2.5 M in hexanes, 22.6 mL, 55.4 mmol) was added to asuspension of ethyl triphenylphosonium iodide (23.2 g, 55.4 mmol) in THF(100 mL) at 25° C. After 30 min, the clear red solution was transferreddropwise by syringe to a vigorously stirred solution of 12 (14.1 g, 55.4mmol) in THF (1100 mL) at −78° C. After addition of the Wittig reagentwas completed, the resulting pale yellow suspension was stirred rapidlyand warmed to 20° C. Then, NaHMDS (1.0 M soln in THF, 55.4 mL, 55.4mmol) was added dropwise by syringe. During the addition of the NaHMDS,the reaction mixture changed from a yellow-orange slurry and to brightred solution. Aldehyde 18E (6.0 g, 18.5 mmol) was then added in THF.After 30 min, the reaction mixture was poured into hexanes (400 mL) andthen 0.5 mL brine was added. The solution was then passed through a plugof SiO₂ eluting with 2:1 hexanes/Et₂O. The iodide was purified by flashchromatography on SiO₂ eluting with hexanes/ethyl acetate (20:1 to 15:1)to give the vinyl iodide 19E (5.0 g, 10.2 mmol, 50%) as a yellow oil: ¹HNMR (400 MHz, CDCl₃): δ 6.95 (s, 1H), 6.50 (s, 1H), 5.45 (dt, J=1.5, 6.8Hz, 1H), 4.22 (t, J=6.4 Hz, 1H), 2.73 (s, 3H), 2.48 (s, 3H), 2.39 (m,2H), 2.02 (s, 3H), 0.90 (s, 9H), 0.06 (3, s), 0.02 (3, s); ¹³C NMR (100MHz, CDCl₃): δ 164.86, 153.46, 142.17, 132.54, 119.23; 115.68, 102.79,77.70, 44.40, 39.09, 26.35, 19.65, 18.63, 14.54, −4.59, −4.84; IR (neat)2928, 1470, 1252, 1068 cm⁻¹.

EXAMPLE 78

[0413] Post-Suzuki, C-15 Hydroxy Tricarbonyl (10E).

[0414] 9-BBN (0.5 M soln in THF, 14.1 mL, 7.03 mmol) was added over a 45min period to a solution of the olefin 7E (2.78 g, 5.41 mmol) in THF (25mL) at 25° C. After 2 h, TLC analysis revealed the complete consumptionof the starting olefin.

[0415] In a separate flask, containing the vinyl iodide 18E (2.65 g,5.41 mmol) and DMF (45 mL), were added successively and with vigorousstirring: Cs₂CO₃ (3.52 g, 10.82 mmol);

[0416] Pd(dppf)₂Cl₂ (1.10 g, 1.35 mmol), AsPh₃ (0.41 g, 1.35 mmol) andH₂O (3.5 mL, 0.19 mol). Then the borane solution, prepared above, wasadded rapidly by syringe to the vigorously stirred solution containingthe vinyl ioidide. After 2 h, the reaction TLC analysis revealed thatthe reaction was complete. The reaction mixture was poured into Et₂O(3×200 mL), brine (1×50 mL) dried over anhydrous MgSO₄. This crudeproduct was purified by flash column chromatography on SiO₂ eluting withhexanes/ethyl acetate (18:1 to 13:1 to 10:1) to afford the TBS protectedcoupled product 9E as an impure mixture which was taken on to the nextstep without further purification.

[0417] The crude TBS protected coupled product 9E was dissolved in 0.5 MHCl in MeOH (30 mL) at 25° C. The reaction was monitored by TLC forcorruption and after 3.5 h (disappearance of starting TBS ether), themixture was poured into a solution of saturated aqueous NaHCO₃ andextracted with CHCl₃ (4×60 mL). The combined organic layers were washedonce with brine (50 mL) and dried over with anhydrous MgSO₄. The diolwas purified by flash column chromatography on SiO₂ eluting withhexanes/ethyl acetate (4:1 to 3:1 to 2:1) to give the pure product 10Eas a clear oil (2.44 g, 3.35 mmol, 62% for two steps): ¹H NMR (400 MHz,CDCl₃: δ 6.96 (s, 1H), 6.56 (s, 1H), 5.16 (t, J=6.9 Hz, 1H), 4.83 (d,J=11.9 Hz, 1H), 4.75 (dd, J=3.4, 8.0 Hz, 1H), 4.70 (d, J=11.9 Hz, 1H),(t, J=6.4 Hz, 1H), 3.45 (q, J=13.2 Hz, 2H), 3.32 (m, 1H), 2.72 (s, 3H),2.32 (t, J=6.5 Hz, 2H), 2.04 (s, 3H), 2.01 (m, 2H), 1.74 (m, 2H), 1.69(s, 3H), 1.45 (s, 9H), 1.38 (s, 6H), 1.09 (d, J=6.9 Hz, 3H), 0.93 (d,J=6.9 Hz, 3H); ¹³C NMR (100 MHz CDCl₃: δ 209.51, 203.04, 166.15, 164.39,154.14, 152.72, 141.71, 138.24, 120.70, 118.76, 115.28, 94.54, 81.85,77.31, 76.57, 63.41, 54.16, 46.47, 41.48, 34.56, 33.95, 31.98, 31.53,27.85, 24.85, 23.45, 21.47, 20.75, 19.04, 15.60, 14.33, 11.35; IR (neat)3546, 3395, 1756, 1717, 1699, 1644, 1621,1506, 1456, 1251, cm⁻¹.

EXAMPLE 79

[0418] Noyori C-31C-15 Diol Product (11E).

[0419] The diketone 10E (1.77 g, 2.43 mmol) was dissolved in 0.12 N HClin MeOH (21 mL, 1.3 eq) at 25° C. The (R)-RuBINAP catalyst (0.045) M inTHF, 8.0 mL, 0.36 mmol) was then added and the mixture transferred to aParr apparatus.

[0420] The vessel was purged with H₂ for 5 min and then pressurized to1200 psi. After 12-14 h at 25° C., the reaction was returned toatmospheric pressure and poured into a saturated solution of NaHCO₃.This mixture was extracted with CHCl₃ (4×50 mL) and the combined organiclayers were dried over anhydrous MgSO₄. The product was purified byflash column chromatography on silica gel eluting with hexanes/ethylacetate (4:1 to 2:1) to give 1.42 g (81%) of the hydroxy ester 11E as agreen foam; ¹H NMR (400 MHz, CDCl₃: δ 6.96 (s, 1H), 6.55 (s, 1H), 5.15(t, J=6.9 Hz, 1H), 4.85 (t, J=5.3 Hz, 1H), 4.81 (d, J=12.0 Hz, 1H), 4.71(d, J=12.0 Hz, 1H), 4.12 (m, 2H), 3.43 (m, 2H), 2.70 (s, 3H), 2.37 (dd,J=2.2, 6.2 Hz, 1H), 2.30 (t, J=6.7 Hz, 2H), 2.24 (dd, J=10.6, 16.2 Hz,1H), 2.03 (s, 3H), 1.99 (m, 2H), 1.68 (S, 3 h), 1.44 (s, 9 h), 1.18 (s,3 h), 1.16 (s, 3 h), 1.09 (d, J=6.8 Hz, 3H), 0.94 (d, J=6.8 Hz, 3H);

[0421]¹³C NMR (100 MHz CDCl₃): δ 215.95, 172.39, 164.39, 154.21, 152.74,141.70, 138.33, 120.59, 118.77, 115.27, 94.64, 82.98, 81.26, 76.51,72.78, 51.82, 41.40, 37.36, 34.66, 33.96, 32.08, 31.10, 30.20, 27.96,25.06, 23.45, 21.73, 21.07, 19.17, 19.01, 16.12, 15.16, 14.33, 12.17; IR(neat) 3434.0, 1757.5, 1704.5, 1249.9, 1152.8 cm⁻¹.

[0422] C-3/C-15 Bis(TES) Carboxylic Acid.

[0423] 2,6-Lutidine (2.1 g, 19.6 mmol) and TESOTf (2.6 g, 9.8 mmol) wereadded successively to a cooled solution of the diol 11E (2.38, 3.26mmol) in CH₂Cl₂ (30 mL) at −78° C. The reaction mixture was stirred at−78° C. for 5 min and then warmed to rt and stirred for 1 hr. Then2,6-lutidine (4.9 g, 45.6 mmol) and TESOTF (6.0 g, 22.8 mmol) were addedsuccessively to a −78° C. cooled solution. The reaction was stirred atrt for 6 hr and then quenched with saturated aqueous NH₄Cl and subjectedto an aqueous workup. The crude product was concentrated in vacuo andthe 2,6-lutidine removed on high vacuum pump and then subjected directlyto the next set of reaction conditions; ¹H NMR (400 MHz, CDCl₃): δ 6.96(s, 1H), 6.66 (s, 1H), 5.04 (t, J=6.93 Hz, 1H), 4.90 (d, J=12.0 Hz, 1H),4.77 (dd, J=7.99, 3.21 Hz, 1H), 4.66 (d, J=12.0 Hz, 1H), 4.46 (m, 1H),4.10 (dq, J=12.3, 7.11 Hz, 2H), 3.42 (m, 1H), 2.70 (s, 3H), 2.60 (dd,J=16.7, 2.34 Hz, 1H), 2.34 (dd, J=16.7, 7.94 Hz, 1H), 2.27 (dd, J=14.0,6.97 Hz, 1H), 2.18 (m, 1H), 2.09 (m, 1H), 2.04 (s, 1H), 1.95 (s, 3H),1.82 (m, 2H), 1.61 (s, 3H), 1.44 (m, 2H), 1.27-1.22 (m, 4H), 1.14 (d,J=8.45 Hz, 3H), 1.11 (d, J=6.81 Hz, 2H), 1.04 (d, J=6.88 Hz, 2H),1.15-1.01 (m, 2H), 0.94 (t, J=7.92 Hz, 18H), 0.65-0.57 (m, 12H); ¹³C NMR(100 MHz, CDCl₃): δ 215.11, 175.34, 165.00, 154.14, 152.80, 142.60,136.84, 121.31, 118.79, 114.60, 94.77, 81.60, 79.06, 76.64, 73.87,54.19, 41.18, 39.56, 35.09, 34.52, 32.29, 31.95, 24.76, 23.62, 22.55,18.95, 18.64, 15.87, 13.69, 11.33, 6.94, 6.83, 5.07, 4.76; IR (neat)3100-2390, 1756.8, 1708.8, 1459.3, 1250.6, 816.1 cm⁻¹.

EXAMPLE 80

[0424] C-15 Hydroxy Acid for Macrolactonization (12E).

[0425] The crude bis(triethylsilyl)ether, prepared above, was dissolvedin 20 mL of dry THF and then cooled to 0° C. Then, 6 ml of 0.12 MHCl/MeOH was added. The reaction mixture was stirred at 0° C. for 3 minand maintained at 0° C. for the duration. The reaction was monitoredclosely by TLC analysis. Methanolic HCl (0.12 M) was added in smallportions, and roughly 1.3 equivalents of 0.12 M HCl was required for thehydrolysis of the C-15 TBS ether (approximately 30-40 mL). The reactionwas complete in appoximately 30 min. The reaction was quenched bypouring into a solution of saturated aqueous NaHCO₃ and subjected to anaqueous workup. Flash column chromatography with 40% EtOAc/hexanesafforded the desired carboxylic acid 12E (1.71 g, 2.20 mmol) in 67%yield; ¹H NMR (400 MHz, CDCl₃): δ 6.96 (s, 1H), 6.69 (1, s), 5.11 (t,J=6.9 Hz, 1H), 4.91 (d, J=12.0 Hz, 1H), 4.71 (dd, J=3.1, 8.2 Hz, 1H),4.64 (d, J=12.0 Hz, 1H), 4.42 (d, J=5.9 Hz, 1H), 4.10 (m, 1H), 3.43 (m,1H), 2.71 (s, 3H), 2.57 (dd, J=2.1, 10.5 Hz, 1H), 2.25 (m, 3H), 2.11 (m,1H), 1.98 (s, 3H), 1.95 (m, 2H), 1.72 (m, 1), 1.67 (s, 3H), 1.45 (m,2H), 1.16 (s, 3H), 1.13 (s, 3H), 1.09 (d, J=6.7 Hz, 3H), 0.99 (d, J=6.7Hz, 3H), 0.95 (t, J=7.9 Hz, 9H), 0.64 (dq, J=2.3, 7.9 Hz, 6H); ¹³C NMR(100 MHz, CDCl₃): δ 215.11, 176.00 (165.10, 154.18, 152.35, 142.24,138.55, 120.74, 118.21, 115.02, 94.76, 81.91, 76.86, 76.63, 73.95,54.08, 41.28, 39.64, 34.73, 34.16, 32.02, 31.67, 24.71, 23.41, 22.49,19.17, 18.62, 15.71, 14.86, 11.20, 6.93, 5.05); IR (neat) 3400-2390,1755.9, 1703.8, 1250.4, 735.4 cm⁻¹.

EXAMPLE 81

[0426] C-3 TriethylsilyllC-7 Trichloroethoxyethyl carbonateMacrolactonizati on Product (13E).

[0427] Triethylamine (155 mg, 1.53 mmol) and 2,4,6-trichlorobenzoylchloride (312 mg, 1.28 mmol) were added to a solution of the hydroxyacid 12E (198 mg, 0.256 mmol) in 3.6 mL of dry THF. The reaction mixturewas stirred for 15 min (and NO LONGER) at rt and then diluted with 20 mLof dry toluene. The resultant solution was added slowly dropwise, viasyringe pump, over 3 hr to a previously prepared, stirred solution ofDMAP (328 mg, 2.68 mmol) in 300 mL of dry toluene. After the addition ofthe substrate was complete, the reaction was stirred for an additional0.5 h and then concentrated in vacuo. Flash column chromatography of thecrude product with 10% EtOAc/hexanes afforded the macrolactone 13E (153mg, 0.20 mmol) in 78% yield; ¹H NMR (400 MHz, CDCl₃): δ 6.96 (s, 1H),6.53 (s, 1H), 5.20 (m, 2H), 5.04 (d, J=10.2 Hz, 1H), 4.84 (d, J=12.0 Hz,1H), 4.78 (d, J=12.0 Hz, 1H), 4.07 (m, 1H), 3.32 (m, 1H), 2.86-2.63 (m,3H), 2.70 (s, 3H), 2.48 (m, 1H), 2.11 (s, 3H), 2.04 (dd, J=6.17, 14.7Hz, 1H), 1.73 (m, 4H), 1.66 (s, 3H), 1.25 (m, 2H), 1.19 (s, 3H), 1.15(s, 3H), 1.12 (d, J=6.68 Hz, 3H), 1.01 (d, J=6.83 Hz, 3H), 0.89 (t,J=8.00 Hz, 9H), 0.58 (q, J=7.83 Hz, 6H); ³C NMR (100 MHz, CDCl₃): δ212.75, 170.66, 164.62, 154.60, 152.52, 140.29, 138.44, 119.81, 119.38,116.28, 94.84, 86.44, 80.14, 76.59, 76.10, 53.55, 45.89, 39.23, 35.47,32.39, 31.69, 31.57, 31.16, 29.68, 27.41, 25.00, 23.44, 22.94, 19.23,18.66, 16.28, 14.83, 6.89, 5.22; IR (neat) 1760.5, 1742.6, 1698.0,1378.8, 1246.2, 1106.0, 729.8 cm⁻¹.

EXAMPLE 82

[0428] SmI₂ Mediated Deprotection of Troc Group.

[0429] Samarium metal (0.52 g, 3.43 mmol) and iodine (0.78 g, 3.09 mmol)in 40 mL of dry, deoxygenated THF were stirred together vigorously atreflux for 2.5 hr. During this period of time, the reaction mixtureprogressed from a dark orange to an olive green to deep blue color. Theresultant deep blue solution of SmI₂ was used directly in the followingreaction. A catalytic amount of NiI₂ (10 mg) was added in one portion tothe vigorously stirted solution of Sm1₂. The reaction mixture wasstirred 5 min at rt and then cooled to −78° C. in a a dry ice/acetonebath. Then, the macrolactone 13E (297 mg, 0.386 mmol), in 10 mL of dryTHF, was added over 1 min to the rapidly stirred, cold solution of 5ml₂/NiI₂. The resultant deep blue solution was maintained at −78° C.with continued vigorous stirring for 1 hr. TLC analysis at this timerevealed the complete consumption of the starting material and formationof a single, lower Rf product. The reaction mixture was quenched withsaturated aqueous NaHCO₃ and subjected to an aqueous workup. Flashcolumn chromatography with 25% EtOAc/hexanes afforded the C-7 alcohol(204 mg, 0.343 mmol) in 89% yield; ¹H NMR (400 MHz, CDCl₃): δ 6.95 (s,1H), 6.54 (s, 1H), 5.15 (m, 1H), 5.05 (d, J=10.15 Hz, 1H), 4.08 (dd,J=10.1, 2.66 Hz, 1H), 3.87 (m,₁H), 3.01 (s, 1H), 3.06 (m, 1H), 2.83-2.65(m, 3H), 2.70 (s, 3H), 2.44 (m, 1H), 2.10 (s, 3H), 2.07 (m, 1H), 1.83(m, 1H), 1.77 (m, 1H), 1.71 (m, 1H), 1.64 (s, 3H), 1.60 (s, 1H), 1.37(m, 1H), 1.31 (m, 1H), 1.20 (m, 1H), 1.15 (s, 3H), 1.14 (m, 5H), 1.02(d, J=7.02 Hz, 3H), 0.89 (t, J=7.97 Hz, 9H), 0.64-0.52 (m, 6H); ¹³C NMR(100 MHz, CDCl₃): δ 218.34, 170.73, 164.59, 152.46, 139.07, 138.49,120.48, 119.54, 116.00, 79.31, 75.81, 73.48, 53.62, 42.98, 39.48, 39.01,32.85, 32.41, 31.20, 26.12, 24.26, 22.01, 22.46, 19.18, 16.44, 15.30,13.99, 6.98 (3), 5.27(3); IR (neat) 3524.0, 1740.3, 1693.4, 1457.2,1378.4, 733.2 cm⁻¹.

EXAMPLE 83

[0430] Desoxyepothilone B (12E).

[0431] The C-3 TES protected alcohol (204 mg, 0.343 mmol) was dissolvedin 6 mL of dry THF in a plastic reaction vessel and cooled to 0° C. inan ice bath. The resultant solution was treated with 3 mL ofHF-pyridine. The reaction mixture was stirred for 80 min at 0° C. andthen quenched by pouring into a saturated aqueous solution of NaHCO₃. Anaqueous workup followed by flash column chromatography with 10%EtOAc/hexanes afforded desoxyepothilone B 12E (160 mg, 0.32 mmol) in950% yield. The resultant product exhibited a ¹H NMR spectrum identicalto desoxyepothilone B prepared as described hereinabove.

[0432] Discussion

[0433] Total Synthesis of (−)-Epothilone A.

[0434] The first known method for preparing epothilone A (1) is providedby this invention. Carbons 9 through 11 insulate domains of chiralityembracing carbons 3 through 8 on the acyl side of the macrolactone, andcarbons 12 through 15 on the alkyl side. Transmitting stereochemicalinformation from one of the segments to the other is unlikely. Thus, theapproach taken deals with the stereochemistry of each segmentindividually. In the acyl segment, this strategy required knowledge ofboth the relative and absolute configurations of the“polypropionate-like” network. In the alkyl segment, two possibilitiesemerge. In one instance, the Cl2-C13 epoxide would be included in theconstruct undergoing merger with the acyl related substructure. In thatcase it would be necessary to secure the relative stereochemicalrelationship of carbons 15, 13 and 12. It was necessary to consider thethe possibility that the epoxide would be deleted from the alkyl-sidemoiety undergoing coupling. This approach would only be feasible if theepoxide could be introduced with acceptable stereocontrol after closureof the macrocycle. The synthesis of compound 4, which contains most ofthe requisite stereochemical information required for the acyl fragment,is described above. This intermediate is prepared by a novel oxidativelyinduced solvolytic cleavage of the cyclopropanopyran 3. Also describedabove is a construct containing the alkyl side coupling partnerembodying the absolute and relative stereochemistry at carbons 15, 13and 12, which differs from the alternative approach set forth below.

[0435] In considering the union of the alkyl and acyl domains, severalpotential connection sites were available. At some point, an acylationwould be required to establish an ester (or lactone) bond (see boldarrow 2). Furthermore, an aldol construction was required to fashion aC₂-C₃ connection. Determining the exact timing of this aldol steprequired study. It could be considered in the context of elongating theC₃-C₉ construct to prepare it for acylation of the C-15 hydroxyl.Unexpectedly, it was discovered that the macrolide could be closed by anunprecedented macroaldolization. (For a previous instance of a ketoaldehyde macroaldolization, see: C. M. Hayward, et al., J. Am. Chem.Soc., 1993, 115, 9345.) This option is implied by bold arrow 3 in FIG.1(A).

[0436] The first stage merger of the acyl and alkyl fragments (see boldarrow 1) posed a difficult synthetic hurdle. It is recognized in the art(P. Bertinato, et al., J. Org. Chem., 1996, 61, 8000; vide infra) thatsignificant resistance is encountered in attempting to accomplish bondformation between carbons 9 and 10 or between carbons 10 and 11, whereinthe epoxide would be included in the alkyl coupling partner. Thesecomplications arose from unanticipated difficulties in fashioning acyland alkyl reactants with the appropriate complementarity for mergeracross either of these bonds. An initial merger between carbons 11 and12 was examined. This approach dictated deletion of the oxirane linkagefrom the O-alkyl coupling partner. After testing several permutations,generalized systems 5 and 6 were examined to enter the first stagecoupling reaction. The former series was to be derived from intermediate4. A de novo synthesis of a usable substrate corresponding togeneralized system 5 would be necessary (FIG. 1(B)).

[0437] The steps leading from 4 to 11 are shown in Scheme 2. Protectionof the future C-7 alcohol (see compound 7) was followed by cleavage ofthe benzyl ether and oxidation to aldehyde 8. Elongation of the aldehydeto the terminal allyl containing fragment 10 proceeded through end ether9 (mixture of E and Z geometrical isomers). Finally, the dithianelinkage was oxidatively cleaved under solvolytic trapping conditions,giving rise to specific coupling component 11. G. Stork; K. Zhao,Tetrahedron Lett. 1989, 30, 287.

[0438] The synthesis of the alkyl fragment started with commerciallyavailable (R)-glycidol 12 which was converted, via its THP derivative13, to alcohol 14. After cleavage of the tetrahydropyran blocking group,the resultant alcohol was smoothly converted to the methyl ketone 15, asshown. The latter underwent an Emmons-type homologation with phosphineoxide 16. D. Meng et al., J. Org. Chem., 1996, 61, 7998. This Emmonscoupling provided a ca. 8:1 mixture of olefin stereoismoers in favor oftrans-17. The resultant alkyne 17 was then converted, via compound 18 toZ-iodoalkene 19 (see FIG. 4(A)). E. J. Corey et al., J. Am.

[0439] Chem. Soc., 1985, 107, 713.

[0440] The critical first stage coupling of the two fragments wasachieved by a B-alkyl Suzuki carbon-carbon bond construction. N. Miyauraet al., J. Am. Chem. Soc., 1989, 111, 314; N.

[0441] Miyaura and A. Suzuki, Chem. Rev., 1995, 95, 2457. Thus,hydroboration of the pre-acyl fragment 11 was accomplished by itsreaction with 9-BBN. The resultant mixed borane cross-coupled toiodoolefin 19, under the conditions indicated, to give 20 in 71% yield.(FIG. 4(B)) Upon cleavage of the acetal, aldehyde 21 was in hand.

[0442] The availability of 21 permitted exploration of the strategy inwhich the methyl group of the C-1 bound acetoxy function would serve asthe nucleophilic component in a macroaldolization. Cf. C. M. Hayward etal., supra. Deprotonation was thereby accomplished with potassiumhexamethyldisilazide in THF at −78° C. Unexpectedly, these conditionsgive rise to a highly stereoselective macroaldolization, resulting inthe formation of the C-3 (S)-alcohol 22, as shown. The heavypreponderance of 22 was favored when its precursor potassium aldolate isquenched at ca. 0° C. When the aldolate was protonated at lowertemperature, higher amounts of the C-3 (R) compound were detected. Infact, under some treatments, the C-3 (R) epimer predominates. It istherefore possible to generate highly favorable C-3(R):C-3(S) ratios inanalytical scale quenches. In preparative scale experiments, the ratioof 22 to its C-3 epimer is 6:1.

[0443] With compound 22 in ready supply, the subgoal of obtainingdesoxyepothilone (23) was feasible. This objective was accomplished byselective removal of the triphenylsilyl (TPS) group in 22, followed,sequentially, by selective silylation of the C-3 alcohol, oxidation ofthe C-5 alcohol, and, finally, fluoride-induced cleavage of the twosilyl ethers.

[0444] Examination of a model made possible bythe published crystalstructure of epothilone (Höfle et al., supra), suggested that theoxirane is disposed on the convex periphery of the macrolide. Oxidationof 23 was carried out with dimethyl dioxirane under the conditionsshown. The major product of this reaction was (−)epothilone A (1), theidentity of which was established by nmr, infrared, mass spectral,optical rotation and chromotaraphic comparisons with authentic material.Höfle et al., supra. In addition to epothilone A (1), small amounts of adiepoxide mixture, as well as traces of the diastereomeric cis C12-C13monoepoxide (>20:1) were detected.

[0445] The method of synthesis disclosed herein provides workable,practical amounts of epothilone A. More importantly, it provides routesto congeners, analogues and derivatives not available from the naturalproduct itself.

[0446] Studies Toward a Synthesis of Epothilone A: Use of HydropyranTemplates for the Management of Acyclic Stereochemical Relationships.

[0447] The synthesis of an enantiomerically pure equivalent of thealkoxy segment (carbons 9-15) was carried out in model studies. The keyprinciple involves transference of stereochemical bias from an(S)-lactaldehyde derivative to an emerging dihydropyrone. The latter, onaddition of the thiazole moiety and disassembly, provides the desiredacyclic fragment in enantiomerically pure form.

[0448] Various novel structural features of the epothilones make theirsynthesis challenging.

[0449] The presence of a thiazole moiety, as well as a cis epoxide, anda geminal dimethyl grouping are key problems to be overcome. Anintriguing feature is the array of three contiguous methylene groupswhich serves to insulate the two functional domains of the molecules.The need to encompass such an achiral “spacer element” actuallycomplicates prospects for continuous chirality transfer and seems tocall for a strategy of merging two stereochemically committedsubstructures. The present invention provides a synthesis of compound 4A(FIG. 14), expecting that, in principle, such a structure could beconverted to the epothilones themselves, and to related screeningcandidates.

[0450] The identification of compound 4A as a synthetic intermediateserved as an opportunity to illustrate the power of hydropyran matricesin addressing problems associated with the control of stereochemistry inacyclic intermediates. The synthesis of dihydropyrones was previouslydisclosed through what amounts to overall cyclocondensation of suitablyactive dienes and aldehydic heterodienophiles. Danishefsky, S. J.Aldrichimica Acta, 1986, 19, 59. High margins of steroselectivity can berealized in assembling (cf. 5A+6A→7A) such matrices (FIG. 13). Moreover,the hydropyran platforms service various stereospecific reactions (seeformalism 7A→8A). Furthermore, the products of these reactions areamenable to ring opening schemes, resulting in the expression of acyciicfragments with defined stereochemical relationships (cf. 8A→9A).Danishefsky, S. J. Chemtracts, 1989, 2, 273.

[0451] The present invention provides the application of two such routesfor the synthesis of compound 4A. Route 1, which does not per se involvecontrol of the issue of absolute configuration, commences with the knownaldehyde 10A. Shafiee, A., et al., J. Heterocyclic Chem., 1979, 16,1563; Schafiee, A.; Shahocini, S. J. Heterocyclic Chem., 1989, 26, 1627.Homologation, as shown, provided enal 12A. Cyclocondensation of 12A withthe known diene (Danishefsky, S. J.; Kitahara, T. J. Am. Chem. Soc.,1974, 96, 7807), under BF₃ catalysis, led to racemic dihydropyrone 13A.Reduction of 13A under Luche conditions provided compound 14A. Luche, J.-L. J. Am. Chem. Soc., 1978, 100, 2226. At this point it was feasible totake advantage of a previously introduced lipase methodology forresolution of glycal derivatives through enzymatically mediated kineticresolution. Berkowitz, D. B. and Danishefsky, S. J. Tetrahedron Lett.,1991, 32, 5497; Berkowitz, D. B.; Danishefsky, S. J.; Schulte, G. K. J.Am. Chem. Soc., 1992, 114,4518. Thus, carbinol 14A was subjected tolipase 30, in the presence of isopropenyl acetate, following theprescriptions of Wong (Hsu, S. -H., et al., Tetrahedron Lett., 1990, 31,6403) to provide acetate 15A in addition to the enantiomerically relatedfree glycal 16A. Compound 15A was further advanced to the PMB protectedsystem 17A. At this juncture, it was possible to use another reactiontype previously demonstrated by the present inventors. Thus, reaction of17A with dimethyldioxirane (Danishefsky, S. J.; Bilodeau, M. T. Angew.Chem. Int. Ed. Engl., 1996, 35, 1381) generated an intermediate(presumably the corresponding glycal epoxide) which, upon treatment withsodium metaperiodate gave rise to aldehyde formate 18A. Allylation of18A resulted in the formation of carbinol 19A in which the formate esterhad nicely survived. (For a review of allylations, see: Yamamoto, Y.;Asao, N. Chem. Rev. 1993, 93, 2207.) However, 19A was accompanied by itsanti stereoisomer (not shown here) [4: 1]. Mesylation of the secondaryalcohol, followed by deprotection (see 19A→20A) and cyclization, asindicated, gave compound 4A.

[0452] In this synthesis, only about half of the dihydropyrone wassecured through the process of kinetic resolution. While, in theory,several of the synthetic stratagems considered contemplate use of eachenantiomer of 15A to reach epothilone itself, another route was soughtto allow for full enantiomeric convergence. The logic of this route isthat the chirality of a “dummy” asymmetric center is communicated to theemerging pyran following previously established principles of tunablediastereoselection in the cyclocondensation reaction. (Danishefsky,supra) Cyclo-condensation of lactaldehyde derivative 21A (Heathcock, C.H., et al., J. Org. Chem., 1980, 45, 3846) with the indicated diene,under ostensible chelation control, afforded 22A. The side chain ethercould then be converted to the methyl ketone 25A as shown (see22A→23A→24A→25A). Finally, an Emmons condensations (for example, see:Lythgoe, B., et al., Tetrahedron Lett., 1975, 3863; Toh, H. T.; Okamura,W. H. J. Org. Chem., 1983, 48, 1414; Baggiolini, E. G., et al., J. Org.Chem., 1986, 51, 3098) of 25A with the phoshphine oxide 26A wastransformed to phosphine oxide 26A according to the procedure describedin Toh, supra) as shown in FIG. 15 gave rise to 27A. (The known2-methyl-4-chloromethylthiazole (see Marzoni, G. J. Heterocyclic Chem.,1986, 23, 577.) A straightforward protecting group adjustment thenafforded the previously encountered 17A. This route illustrates theconcept of stereochemical imprinting through a carbon center whicheventually emerges in planar form after conferring enantioselection tosubsequently derived stereocenters. The use of the dihydropyrone basedlogic for securing the stereochemical elements of the epothilones, aswell as the identification of a possible strategy for macrocyclizationwill be described in the following section.

[0453] Studies Toward a Synthesis of Epothilone A: Sterocontrolled

[0454] Assembly of the Acyl Region and Models for Macrocyclization.

[0455] Ring-forming olefin metathesis has been employed to construct16-membered ring congeners related to epothilone A. A stereospecificsynthesis of the C₃-C₉ sector of the acyl fragment was achieved byexploiting a novel oxidative opening of a cyclopropanated glycal.

[0456] Disclosed in the previous section is a synthesis of the “alkoxy”segment of epothilone (1) (see compound 2B, FIG. 7) encompassing carbons10 to 21. In this section the synthesis of another fragment encoding thestereochemical information of acyl section carbons 3 to 9. It wasenvisioned that the aldehydo center (C₃) of the formal target 3B wouldserve as an attachment site to a nucleophilic construct derived fromcompound 2B (requiring placement of a 2 carbon insert, as suggested inFIG. 7), through either inter- or intramolecular means. In such acontext, it would be necessary to deal independently with thestereochemistry of the secondary alcohol center eventually required atC₃. One of the interesting features of system 3B is the presence ofgeminal methyl groups at carbon 4 (epothilone numbering). Again, use ismade of a dihydropyran strategy to assemble a cyclic matrixcorresponding, after appropriate disassembly, to a viable equivalent ofsystem 3B. The expectation was to enlarge upon the dihydropyran paradigmto include the synthesis of gem-dimethyl containing cyclic and acyclicfragments. The particular reaction type for this purpose is generalizedunder the heading of transformation of 4B -5B (see FIG. 7). Commitmentas to the nature of the electrophile E is avoided. Accordingly, thequestion whether a reduction would or would not be necessary in goingfrom structure type 5B to reach the intended generalized target 3B isnot addressed.

[0457] The opening step consisted of a stereochemically tuneable versionof the diene-aldehyde cyclocondensation reaction (FIG. 8; Danishefsky,S. J., Aldrichimica Acta, 1986, 19, 59), in this instance drawing uponchelation control in the merger of the readily availableenantiomerically homogenous aldehyde 6B with the previously known diene7B. Danishefsky, S. J., et al., J. Am. Chem. Soc. 1979, 101, 7001.Indeed, as precedent would have it, under the influence of titaniumtetrachloride there was produced substantially a single isomer shown ascompound 8B. In the usual and stereochemically reliable way(Danishefsky, S. J., Chemtracts Org. Chem. 1989, 2, 273), thedihydropyrone was reduced to the corresponding glycal, 9B. At thispoint, it was feasible to utilize a directed Simmons-Smith reaction forthe conversion of glycal 9B to cyclopropane 10B. Winstein, S.;Sonnenberg, J. J. Am. Chem. Soc., 1961, 83, 3235; Dauben, W. G.;Berezin, G. H. J. Am. Chem. Soc., 1963, 85, 468; Furukawa, J., et al.,Tetrahedron, 1968, 24, 53; For selected examples, see Soeckman, R. K.Jr.: Charette, A. B.; Asberom, T.; Johnston, B. H. J. Am. Chem. Soc.,1991, 113, 5337; Timmers, C. M.; Leeuwenurgh, M. A.; Verheijen, J. C.;Van der Marel, G. A.; Van Boom, J. H. Tetrahedron: Asymmetry, 1996, 7,49. This compound is indeed an interesting structure in that itcorresponds in one sense to a cyclopropano version of a C-glycoside. Atthe same time, the cyclopropane is part of a cyclopropylcarbinyl alcoholsystem with attendant possibilities for rearrangement. Wenkert, E., etal., J. Amer. Chem. Soc., 1970, 92, 7428. It was intended to cleave theC-glycosidic bond of the cyclopropane in a fashion which would elaboratethe geminal methyl groups, resulting in a solvent-derived glycoside withthe desired aidehyde oxidation state at C-3 (see hypothesizedtransformation 4B→5B, FIG. 7). In early efforts, the non-oxidativeversion of the projected reaction (i.e. E⁺=H⁺) could not be reduced topractice. Instead, products clearly attributable to the ring expandedsystem 11 were identified. For example, exposure of 10B to acidicmethanol gave rise to an epimeric mixture of seven-memberedmixed-acetals, presumably through the addition of methanol tooxocarbenium ion 11B.

[0458] However, the desired sense of cyclopropane opening, under theinfluence of the ring oxygen, was achieved by subjecting compound 10B tooxidative opening with N-iodosuccinimide. (For interestingHg(II)-induced solvolyses of cyclopropanes that are conceptually similarto the conversion of 10B to 12B, see: Collum, D. B.; Still, W. C.;Mohamadi, F. J. Amer. Chem. Soc., 1986, 108, 2094; Collum, D. B.;Mohamadi, F.; Hallock, J. S.; J. Amer. Chem. Soc., 1983, 105, 6882.Following this precedent, a Hg(II)-induced solvolysis of cyclopropane10B was achieved, although this transformation proved to be lessefficient than the reaction shown in FIG. 8.) The intermediateiodomethyl compound, obtained as a methyl glycoside 12B, when exposed tothe action of tri-n-butyltinhydride gave rise to pyran 13B containingthe geminal methyl groups. Protection of this alcohol (see 13B 14B),followed by cleavage of the glycosidic bond, revealed the acyclicdithiane derivative 15B which can serve as a functional version of thehypothetical aldehyde 3B.

[0459] Possible ways of combining fragments relating to 2B and 3B in afashion to reach epothilone and congeners thereof were examined. In viewof the studies of Schrock (Schrock, R. R., et al., J. Am. Chem. Soc.,1990, 112, 3875) and Grubbs (Schwab, P. et al., Angew. Chem. Int. Ed.Engl., 1995, 34, 2039; Grubbs, R. H.; Miller, S. J. Fu, G. C. Acc. Chem.Res., 1995, 28, 446; Schmalz, H. -G., Angew. Chem. Int. Ed. Engl., 1995,34, 1833) and the disclosure of Hoveyda (Houri, A. F., et al., J. Am.Chem. Soc., 1995, 117, 2943), wherein a complex lactam was constructedin a key intramolecular olefin macrocyclization step through amolybdenum mediated intramolecuar olefin in metathesis reaction(Schrock, supra; Schwab, supra), the possibility of realizing such anapproach was considered. (For other examples of ring-closing metathesis,see: Martin, S. F.; Chen, H. -J.; Courtney, A. K.; Lia, Y.; Patzel, M.;Ramser, M N.; Wagman, A. S. Tetrahedron, 1996, 52, 7251; Furstner, A.;Langemann, K. J. Org. Chem., 1996, 61, 3942.)

[0460] The matter was first examined with two model ω-unsaturated acids16B and 17B which were used to acylate alcohol 2B to provide esters 18Band 19B, respectively (see FIG. 9). These compounds did indeed undergoolefin metathesis macrocyclization in the desired manner under theconditions shown. In the case of substrate 18B, compound set 20B wasobtained as a mixture of E- and Z-stereoisomers [ca. 1:1]. Diimidereduction of 20B was then conducted to provide homogeneous 22B. Theolefin methathesis reaction was also extended to compound 19B bearinggeminal methyl groups corresponding to their placement at C4 ofepothilone A. Olefin metathesis occurred, this time curiously producingolefin 21B as a single entity in 70% yield (stereochemisty tentativelyassigned as Z.) Substantially identical results were obtained throughthe use of Schrock's molybdenum alkylidene metathesis catalyst.

[0461] As described above, olefin metathesis is therefore amenable tothe challenge of constructing the sixteen membered ring containing boththe required epoxy and thiazolyl functions of the target system. It ispointed out that no successful olefin metathesis reaction has yet beenrealized from seco-systems bearing a full compliment of functionalityrequired to reach epothilone. These negative outcomes may merely reflecta failure to identify a suitable functional group constraint patternappropriate for macrocylization.

[0462] The Total Synthesis of Epothilone B: Extension of the SuzukiCoupling Method

[0463] The present invention provides the first total synthesis ofepothilone A (1). D. Meng, et al., J. Org. Chem, 1996, 61, 7998 P.Bertinato, et al., J. Org. Chem, 1996, 61, 8000. A. Balog, et al.,Angew. Chem. Int. Ed. Engl., 1996, 35, 2801. D. Meng, et al., J. Amer.Chem. Soc., 1997, 119, 10073. (For a subsequent total synthesis ofepothilone A, see: Z. Yang, et al., Angew. Chem. Int. Ed. Engl., 1997,36, 166.) This synthesis proceeds through the Z-desoxy compound (23)which underwent highly stereoselective epoxidation with2,2-dimethyldioxirane under carefully defined conditions to yield thedesired βepoxide. The same myxobacterium of the genus Sorangium whichproduces 23 also produces epothilone B (2). The latter is a more potentagent than 23, both in antifungal screens and in cytotoxicity/cellnucleus disintegration assays. G. Höfle, et al., Angew. Chem. Int. Ed.Engl. 1996, 35, 1567; D. M. Bollag, et al., Cancer Res. 1995, 55, 2325.

[0464] An initial goal structure was desoxyepothilone B (2C) or asuitable derivative thereof.

[0465] Access to such a compound would enable the study of the regio-and stereoselectivity issues associated with epoxidation of the C12-C13double bond. A key issue was the matter of synthesizingZ-tri-substituted olefinic precursors of 2C with high margins ofstereoselection.

[0466] A synthetic route to the disubstituted system (A. Balog, et al.,Agnew. Chem. Int. Ed. Engl., 1996, 35, 2801) employed apalladium-mediated B-alkyl Suzuki coupling (N. Miyaura, et al., J. Am.Chem. Soc. 1989, 171, 314. (For a review, see: N. Miyaura, A. Suzuki,Chem. Rev. 1995, 95, 2457) of the Z-vinyl iodide 19 (FIG. 4(A)) withborane 7C derived from hydroboration of compound 11 (FIG. 1(A)) with9-BBN (FIG. 4(B)).)

[0467] A preliminary approach was to apply the same line of thinking toreach a Z-tri-substituted olefin (FIG. 17) en route to 2C. Two issueshad to be addressed. First, it would be necessary to devise a method toprepare vinyl iodide 8C, the tri-substituted analog of 19. If this goalcould be accomplished, a question remained as to the feasibility ofconducting the required B-alkyl Suzuki coupling reaction to reach aZ-tri-substituted olefin. The realization of such a transformation witha “B-alkyl” (as opposed to a “B-alkenyl” system) at the inter-molecularlevel, and where the vinyl iodide is not of the β-iodoenoate (orβ-iodoenone) genre, was not precedented. (For some close analogies whichdiffer in important details from the work shown here, see: N. Miyaura,et al., Bull. Chem. Soc. Jpn. 1982, 55, 2221; M. Ohba, et al.,Tetrahedron Lett., 1995, 36, 6101; C. R. Johnson, M. P. Braun, J. Am.Chem. Soc. 1993, 115, 11014.)

[0468] The synthesis of compound 8C is presented in FIG. 16. The routestarted with olefin 10C which was prepared by catalytic asymmetricallylation of 9C (G. E. Keck, et al., J.

[0469] Am. Chem. Soc., 1993, 115, 8467) followed by acetylation.Site-selective dihydroxylation of 10C followed by cleavage of the glycolgenerated the unstable aldehyde 11C. Surprisingly, the latter reactedwith phosphorane 12C (1. Chen, et al., Tetrahedron Lett., 1994, 35,2827) to afford the Z-iodide 8C albeit in modest overalI yield. Borane7C was generated from 11 as described herein. The coupling of compound7C and iodide 8C (FIG. 16) could be conducted to produce the pureZ-olefin 13C.

[0470] With compound 13C in hand, protocols similar to those employed inconnection with the synthesis of 23 could be used. (A. Balog, et al.,Angew. Chem. Int. Ed. Engl., 1996, 35, 2801). Thus, cleavage of theacetal linkage led to aldehyde 14C which was now subjected tomacroaldolization (FIG. 17). The highest yields were obtained bycarrying out the reaction under conditions which apparently equilibratethe C3 hydroxyl group. The 3R isomer was converted to the required 3Sepimer via reduction of its derived C3-ketone (see compound 15C). Thekinetically controlled aldol condensation leading to the natural 35configuration as discribed in the epothilone A series was accomplished.However, the overall yield for reaching the 3S epimer is better usingthis protocol. Cleavage of the C-5 triphenylsilyl ether was followedsequentially by monoprotection (t-butyldimethylsilyl) of the C3hydroxyl, oxidation at C5 (see compound 16C), and, finally, cleavage ofthe silyl protecting groups to expose the C3 and C7 alcohols (seecompound 2C).

[0471] It was found that Z-desoxyepothilone B (2C) undergoes very rapidand substantially regio- and stereoselective epoxidation under theconditions indicated (although precise comparisons are not available,the epoxidation of 2C appears to be more rapid and regioselective thanis the case with 23) (A. Balog, et al., Angew. Chem. Int. Ed. Engl.,1996, 35, 2801), to afford epothilone B (2) identical with an authenticsample (¹H NMR, mass spec, IR, [α]_(D)). Accordingly, the presentinvention dislcoses the first total synthesis of epothilone B. Importantpreparative features of the present method include the enantioselectivesynthesis of the trisubstituted vinyl iodide 8C, the palladium-mediatedstereospecific coupling of compounds 7C and 8C to produce compound 13C(a virtually unprecedented reaction in this form), and the amenabilityof Z-desoxyepothilone B (2C) to undergo regio- and stereoselectiveepoxidation under appropriate conditions.

[0472] Desmethylepothilone A

[0473] Total syntheses of epothilones A and B have not been previouslydisclosed. Balog, A., et al., Angew. Chem., Int. Ed. Engl. 1996, 35,2801; Nicolaou, K. C., et al., Angew. Chem., Int. Ed. Engi. 1997, 36,166. Nicolaou, K. C., et al., Angew. Chem., Int. Ed. Engl. 1997, 36,525; Schinzer, D., et al., Angew. Chem., Int. Ed. Engl. 1997, 36, 523.Su, D. -S., et al., Angew. Chem. Int. Ed. Engl. 1997, 36, 757. The modeof antitumor action of the epothilones closely mimics that of Taxol®(paclitaxel). Höfle, G., et al., H. Angew. Chem., Int. Ed. Engl. 1996,35, 1567. Although Taxol® is a clinically proven drug, its formulationcontinues to be difficult. In addition, Taxol® induces the multidrugresistance (MDR) phenotype. Hence, any novel agent that has the samemechanism of action as Taxol® and has the prospect of having superiortherapeutic activity warrants serious study. Bollag, D. M., et al.,Cancer Res. 1995, 55, 2325.

[0474] The present invention provides epothilone analogs that are moreeffective and more readily synthesized than epothilone A or B. Thesyntheses of the natural products provide ample material for preliminarybiological evaluation, but not for producing adequate amounts for fulldevelopment. One particular area where a structural change could bringsignificant relief from the complexities of the synthesis would be inthe deletion of the C8 methyl group from the polypropionate domain (seetarget system 3D). The need to deal with this C8 chiral centercomplicates all of the syntheses of epothilone disclosed thus far.Deletion of the C8 methyl group prompts a major change in syntheticstrategy related to an earlier diene-aldehyde cyclocondensation route.Danishefsky, S. J. Chemtracts 1989, 2, 273; Meng, D., et al., J. Org.Chem. 1996, 61, 7998; Bertinato, P., et al., J. Org. Chem. 1996, 61,8000.

[0475] As shown in FIG. 20, asymmetric crotylation (87% ee) of 4D(Brown, H. C.; Bhat, K. S. J. Am. Chem. Soc. 1986, 108, 5919), followedby protection led to TBS ether 5D. The double bond was readily cleavedto give aldehyde 6D. The aldehyde was coupled to the dianion derivedfrom t-butyl isobutyrylacetate to provide 7D. The ratio of the C₅₅ (7D):C_(5R) compound (not shown) is ca 10:1. That the Weiler-type β-ketoesterdianion chemistry (Weiler, L. J. Am. Chem. Soc. 1970, 92, 6702.; Weiler,L.; Huckin, S. N. J. Am. Chem. Soc. 1974, 96, 1082) can be conducted inthe context of the isobutyryl group suggested several alternateapproaches for still more concise syntheses. Directed reduction of theC₃ ketone of 7D following literature precedents (Evans, D. A., et al.,J. Org. Chem. 1991, 56, 741), followed by selective silylation of the C₃hydroxyl gave a 50% yield of a 10:1 ratio of the required C3s (seecompound 8D) to C₃R isomer (not shown). Reduction with sodiumborohydride afforded a ca. 1:1 mixture of C₃ epimers. The carbinol,produced upon debenzylation, was oxidized to an aldehyde which,following methylenation through a simple Wittig reaction, affordedolefin 9D. Treatment of this compound with TBSOTf provided ester 10Dwhich was used directly in the Suzuki coupling with the vinyl iodide12D.

[0476] The hydroboration of 10D with 9-BBN produced intermediate lDwhich, on coupling with the vinyl iodide 12D and in situ cleavage of theTBS ester led to 13D (FIG. 21). After deacetylation, the hydroxy acid14D was in hand. Macrolactonization of this compound (Boden, E. P.;Keck, G. E. J. Org. Chem. 1985, 50, 2394) produced 15D which, afterdesilylation, afforded C₈-desmethyldesoxyepothilone (16D). Finally,epoxidation of this compound with dimethyldioxirane produced the goalstructure 3D. The stereoselectivity of epoxidation was surprisingly poor(1.5:1) given that epoxidation of desoxyepothilone A occurred with >20:1stereoselectivity. Deletion of the C₈ methyl group appears to shift theconformational distribution of 16D to forms in which the epoxidation bydimethyl dioxirane is less β-selective. It is undetermined whether theeffect of the C₈ methyl on the stereoselectivity of epoxidation bydimethydioxirane and the dramatic reduction of biological activity arerelated.

[0477] Compounds 3D and 16D were tested for cytotoxicity in cellcultures and assembly of tubulin in the absence of GTP. Microtubuleprotein (MTP) was purified from calf brains by two cycles of temperaturedependent assembly and disassembly. Weisenberg, R. C. Science 1972, 177,1104. In control assembly experiments, MTP (1 mg/mL) was diluted inassembly buffer containing 0.1 M MES (2-(N-morpholino) ethanesulfonicacid), 1 mM EGTA, 0.5 mM MgCl₂, 1 mM GTP and 3M glycerol, pH 6.6. Theconcentration of tubulin in MTP was estimated to be about 85%. Assemblywas monitored spectrophotometrically at 350 nm, 35° C. for 40 min byfollowing changes in turbidity as a measure of polymer mass. Gaskin, F.;Cantor, C. R.; Shelanksi, M. L. J. Mol. Biol. 1974, 89, 737. Drugs weretested at a concentration of 10 μM, in the absence of GTP. Microtubuleformation was verified by electron microscopy. To determine thestability of microtubules assembled in the presence of GTP or drug,turbidity was followed for 40 min after the reaction temperature wasshifted to 4° C.

[0478] Cytotoxicity studies showed drastically reduced activity in the8-desmethyl series. Compounds 3D and 16D were approximately 200 timesless active than their corresponding epothilone A counterparts (seeTable 1). Recalling earlier SAR findings at both C₃ and C₅, inconjunction with the findings disclosed herein, the polypropionatesector of the epothilones emerges as a particularly sensitive locus ofbiological function. Su, D. -S., et al., Angew. Chem. Int. Ed. Engl.1997, 36, 757; Meng, D., et al., J. Am. Chem. Soc. 1997, 119. TABLE 1Relative efficacy of epothilone compounds against drug-sensitive andresistant human leukemic CCRF-CEM cell lines.^(a) CCRF-CEM CCRF-CEM/VBLCCRF-CEM/VM₁ Compound IC₅₀ (μM)^(b) IC₅₀ (μM)^(b) IC₅₀ (μM)^(b) 16D 5.005.75 6.29 3D 0.439 2.47 0.764 epothilone A 0.003 0.020 0.003desoxyepothilone A 0.022 0.012 0.013 epothilone B 0.0004 0.003 0.002desoxyepothilone B 0.009 0.017 0.014 paclitaxel 0.002 3.390 0.002

[0479] Desoxyepothiline B: an Effective Microtubule-targeted AntitumorAgent with a Promising in vivo Profile Relative to Epothilone b

[0480] The epothilones have been synthesized as herein disclosed andevaluated for antitumor potential in vitro and in vivo. Epothilones andpaclitaxel are thought to share similar mechanisms of action instabilizing microtubule arrays as indicated by binding displacementstudies, substitution for Taxol® in Taxol®-dependent cell growth, andelectron microscopic examinations. Cell growth inhibitory effects havebeen determined in two rodent and three human tumor cell lines and theirdrug resistant sublines. While Taxol® showed as much as 1970-foldcross-resistance to the sublines resistant to Taxol®, adriamycin,vinblastine or actinomycin D, most ephothilones exhibit little or nocross-resistance. In multidrug resistant CCRF-CEM/VBL₁₀₀ cells, the 50%cell growth inhibitory concentrations (IC₅1 values) for epothilone A,epothilone B, desoxyepothilone A, desoxy epothilone B and Taxol® were0.02, 0.002, 0.012, 0.017 and 4.14 μM, respectively. In vivo studies,using i.p. administration, indicate that the parent, epothilone B, ishighly toxic to mice with little therapeutic effect when compared withlead compound desoxyepothilone B (25-40 mg/kg, Q2Dx5, i.p.), whichshowed far superior therapeutic effect and lower toxicity thanpaclitaxel, doxorubicin, camptothecin or vinblastine (at maximaltolerated doses) in parallel experiments. In nude mice bearing a humanmammary carcinoma xenograft (MX-1), marked tumor regression and cureshave been obtained with desoxyepothilone B.

[0481] The isolation of the naturally occurring macrolides epothilone Aand epothilone B from the myoxobacteria Sorangium cellulosum (Hoefle,G., et al., Angew. Chem. Int. Ed. Engl. 1996, 35, 567-1569; Gerth, K.,et al. J. Antibiot. 1996, 49, 560-563) and the subsequent demonstrationof their ability to stabilize microtubule arrays in vitro elicitedconsiderable interest in this class of compounds (Bollag, D. M., et al.,Cancer Res. 1995, 55, 2325-2333; Su, D. -S., et al., Angew. Chem. Int.Ed. Engl. 1997, 36, 2093-2096; Meng, D., et al., J. Am. Chem. Soc.1997,119, 2733-2734; Muhlradt, P. F. & Sasse, F. Cancer Res. 1997, 57,3344-3346; Service, R. E. Science 1996, 274, 2009). We have recentlyconducted the total synthesis of these natural products as well as over45 related analogs (Meng, D., et al., J. Am. Chem. Soc. 1997,119,10073-10092; Su, D -S., et al., Angew. Chem. Int. Ed. Engl. 1997,36,757-759; Chou, T. -C., Zhang, X. -G., & Danishefsky, S. J. Proc. Am.Assoc. Cancer Res. 1998, 39, 163-164) in order to investigate theirchemical structure-biological activity relationships (Su, D.-S, et al.,Agnew. Chem. Int. Ed. Engl. 1997, 36, 2093-2096). The studies disclosedherein allowed the characterization of the epothilone structure in threezones. Thus, in the C-1˜8 acyl sector, the present inventors havedetermined that structural changes are not tolerated in terms of invitro cytoxocity and microtubule stabilizing ability. This stands incontrast to the C-9˜15 O-alkyl sector and the C-15 pendant aryl sectorswherein considerable modification of structures is tolerated (Su, D.-S., et al., Angew. Chem. Int. Ed. Engl. 1997, 36, 2093-2096; Meng, D.,et al., 1997, J. Am. Chem. Soc. 119, 10073-10092). Described herein arethe results of in vitro and in vivo experiments on the Z-12,13 desoxyversion of epothilone B (desoxyepothilone B).

[0482] It has been shown that the natural epothilones A and B have asimilar mechanism of action to paclitaxel (Taxol®) although structurallydiverse (Su, D. -S., et al., Angew. Chem. Int. Ed. Engl. 1997, 36,2093-2096; Meng, D., et al., J. Am. Chem. Soc. 1997, 19, 2733-2734;Schiff, P. B., Fant, J. & Horwitz, S. B. Nature 1979, 277, 665-667;Landino, L. M. & MacDonald, T. L., in: The Chemistry and Pharmacology ofTaxol and Its Derivatives, Favin, V., ed., Elsevier, New York 1995,,Chapter 7, p. 301). Paclitaxel, isolated from the Pacific yew tree(Taxus brevifolia), has been widely used clinically to treat a varietyof solid cancers including neoplasms of ovary, breast, colon and lung(Landino, L. M. & MacDonald, T. L. id.; Rose, W. C. Anti-Cancer Drugs,1992, 3, 311-321; Rowinsky, E. K., et al., Seminars Oncol. 1993, 20,1-15). Epothilones A and B as well as Taxol® stabilize microtubuleassemblies as demonstrated by binding displacement, substitution forpaclitaxel in paclitaxel-dependent cell growth, and electron microscopicexaminations (Bollag, D. M., et al., Cancer Res. 1995, 55, 2325-2333).Despite these similarities, the epothilones are more water soluble thanpaclitaxel, thereby offering potentially distinct advantages forformulation. Epothilones are more potent than paclitaxel in inhibitingcell growth, especially against cells expressing P-glycoprotein (Pgp)that are multidrug resistant (MDR), including cross-resistance topaclitaxel (Bollag, D. M., id.; Su, D. -S., et al., Angew. Chem. Int.Ed. Engl. 1997, 36, 2093-2096).

[0483] Materials and Methods

[0484] All stock solutions of the above (except VBL in saline) wereprepared using dimethylsulfoxide (DMSO) as a solvent and were furtherdiluted to desired concentrations for experimental use. The finalconcentration of DMSO in tissue culture was 0.25% (v/v) or less to avoidsolvent cytotoxicity. For in vivo studies, paclitaxel in Cremophor-EtOHwas further diluted with DMSO as needed. Vinblastine sulfate (Velban)(Eli Lilly & Co. Indianapolis, Ind.), and doxorubicin or adriamycin HCl(DX or Adr) (Pharmacia, Columbus, Ohio) in saline were diluted with DMSOas needed. DMSO was used as a vehicle for epothilones. Each mousereceived <40 μL DMSO in all experiments.

[0485] Cell Lines

[0486] The CCRF-CEM human T-cell acute lymphoblastic leukemia cell lineand its vinblastine-resistant (CCRF-CEM/VBL,₁₀₀) andteniposide-resistant (CCRF-CEM/VM₁) sublines (Cass, C. E., et al., 1989,Cancer Res. 49, 5798-5804; Danks, M. K., Yalowich, J. C., & Beck, W. T.1987, Cancer Res. 47, 1297-1301) were used. CCRF-CEM/Taxol® wasdeveloped by the present inventors following continuous exposure ofCCRF-CEM cells with increasing concentrations of paclitaxel (atIC₅₀˜IC₉₀) for ten months. The fresh medium with paclitaxel wasreplenished every week. The CCRF-CEM/Taxol® exhibited 57-fold resistanceto paclitaxel (IC₅₀=0.0021 μM, see Table 1A). The DC-3F hamster lungfibroblast cell line and its actinomycin D-selected sublines (DC-3F/ADIIand DC-3F/ADX) were obtained from the Memorial Sloan-Kettering CancerCenter (MSKCC). The murine leukemic P388/0 and its doxorubicin-selectedsubline (P388/DX) as well as human neuroblastoma SK-N-As and itsdoxorubicin-selected subline (SK-N-FI/Adr) were obtained from MSKCC.

[0487] The drug-resistant cell lines were continuously cultured in thepresence of the selecting agent, AD, DX, VBL or VM to maintain the drugresistant phenotypes. Each sub-cell line was cultured for one to twopassages in an appropriate concentration (e.g. IC₅₀) of the drug, whichwas then removed from the media and the cells were rested in fresh mediafor a minimum of 4 days before each assay. All cells were cultured inRPMI 1640-10% FBS at 37° C., 5% CO₂ (see below).

[0488] Cytotoxicity Assays

[0489] The cells were cultured at an initial density of 5×10⁴ cells/mL.They were maintained in a 5% CO₂-humidified atmosphere at 37° C. inRPMI-1640 medium (GIBCO-BRL, Gaithersburg, Md.) containing penicillin(100U/mL), streptomycin (100 mg/mL) (GIBCO-BRL) and 10% heat inactivatefetal bovine serum. Culture for cell suspension (such as for CCRF-CEM,P388 and sublines), were performed by the XTT-microculture tetrazoniummethod (Scudiero, D. A. et al., Cancer Res. 1988, 48, 4827-4833) induplicate in 96-well microtiter plates.

[0490] 2′, 3′-B is(methoxy-4-nitro-5-sufophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hyudroxide (XTT) was prepared at 1 mg/mLin prewarmed (37° C.) medium without serum. Phenazine methosulfate (PMS)and fresh XTT were mixed together to obtain 0.025 mM PMS-XTT solution(25 μL of the stock 5 mM PMS was added per 5 mL of 1 mg/mL XTT).Following a 72 h incubation, 50 μL of the assay aliquots were added toeach well of the cell culture.

[0491] After incubation at 37° C. for 4 h, absorbance at 450 nm and 630nm was measured with a microplate reader (EL340, Bio-Tek Instruments,Inc., Winooski, Vt.).

[0492] The cytotoxicity of the drug toward the monolayer cell cultures(such as DC-3F, MCF-7, SK-N-As and sublines) was determined in 96-wellmicrotiter plates by the SRB method as described by Skehan andco-workers (Skehan, P., et al., J. Natl. Cancer Inst. 1990, 82,1107-1112) for measuring the cellular protein content. Cultures werefixed with trichloroacetic acid and then stained for 30 min with 0.4%suforhodamine B dissolved in 1% acetic acid.

[0493] Unbound dye was removed by acetic acid washes, and theprotein-bound dye was extracted with an unbuffered Tris base(tris(hydroxy-methyl)aminomethane) for determination of absorbance at570 nm in a 96-well microtiter plate reader. The experiments werecarried out in duplicate. Each run entailed six to seven concentrationsof the tested drugs. Data were analyzed with the median-effect plot(Chou, T. -C. & Talalay, P. T. (1984) Adv. Enzyme Regul. 22, 27-55)using a previously described computer program (Chou, J., & Chou T. -C.1987, Dose-effect analysis with microcomputers: Quantitation of ED ₅₀ ,synergism, antagonism, low-dose risk, reception-ligand binding andenzyme kinetics, IBM-PC software and manual, Biosoft, Cambridge, U.K.).

[0494] Stability of Desoxyepothilone B in Plasma

[0495] H PLC Method.

[0496] Sample Preparation. To 300 microliters of spiked plasma are added30 microliters of methanol. The mixture is agitated and allowed to standfor 2 minutes. Then 600 microliters of methanol are added. The mixtureis centrifuged. The supernatant is removed for analysis by HPLC.Analyses were performed underthe following chrmotographic conditions:Column: Nova-Pak C18, 15 cm. Eluant: 50% acetonitrilelwater with 0.8%triethylamine, 0.2% phosphoric acid. Detection: UV (250 nm).

[0497] Animals

[0498] Athymic nude mice (nu/nu) were used for MX-1 and MCF-7/Adr humanmammary carcinoma xenografts. Mice were obtained from Taconic LaboratoryAnimals and Service (Germantown, N.Y.: outbred, Swiss background). Malemice 6-8 weeks old, weighing 20-25 g were used.

[0499] Results

[0500] Structure-Activity Relationships

[0501] To determine structure-activity relationships of epothilones (Su,D. -S., et al., Angew. Chem. Int. Ed. Engl. 1997, 36, 2093-2096), thesusceptibility of CCRF-CEM leukemic cells and the respectivedrug-resistant sublines CCRF-CEM/VBL₁₀₀ (Pgp-MDR cells) (Cass, C. E., etal., Cancer Res. 1989, 49, 5798-5804) and CCRF/CEM/VM₁ (cells with amutated topo II gene) (Danks, M. K., Yalowich, J. C., & Beck, W. T.Cancer Res. 1987, 47, 1297-1301) to epothilones A and B anddesoxyepthilone B (Table 1A) were determined. Although/VBL,₁₀₀ is527-fold resistant to VBL and 1970-fold resistance to paclitaxel, theepothilones A and B exhibited only 6.1˜7.4-fold resistance, whiledesoxyepothilones A and B evidenced only 0.6˜1.8-fold resistance. Usingpaclitaxel as the selecting agent, CCRF-CEM/Taxol® was grown 57-foldresistant and found to be 10.9-fold resistance to VBL. By contrast, DX,AD and VP-16 showed only 2.3-4.5 fold resistance, and epothilones A andB showed very little resistance (i.e., 1.4-3.1-fold) anddesoxyepothilones A and B displayed almost no resistance (i.e., 0.7˜1.7fold) (Table 1A). CCRF-CEM/VM₁ cells that were 117-fold resistant toetoposide were sensitive to all epothilones or desoxyepothilones listedin Table 1A with only 0.6-3.6 fold resistance. TABLE 1A Susceptibilityof CCRF-CEM and its drug resistant sublines to epothilone derivatives.(C) (A) (B) CCRF- (D) CCRF- CCRF-CEM/ CEM/ CCRF- Compound CEM VBL₁₀₀Taxol ® CEM/VM₁ (B)(A) (C)(A) (D)(A) IC₅₀ (μM)* Epo A 0.0027 0.0200.0037 0.0061 7.4 1.4 2.3 Epo B 0.00035 0.0021 0.0011 0.0013 6.1 3.1 3.6dEpo A 0.0220 0.012 0.0150 0.013 0.55 0.7 0.59 dEpo B 0.0095 0.0170.0162 0.014 1.8 1.7 1.5 Taxol ® 0.0021 4.140 0.120 0.0066 1971 57 3.1Vinblastine 0.0063 0.332 0.0069 0.00041 527 10.9 0.7 Etoposide 0.29010.30 1.32 34.4 35 4.5 117 Adriamycin 0.036 1.74 0.082 0.128 48 2.3 3.6Actinomycin 0.00035 0.038 0.0013 0.00027 109 3.7 0.8 D #antagonism,low-dose risk, reception-ligand binding and enzyme kinetics, 1987,IBM-PC software and manual, Biosoft, Cambridge, U.K.)

[0502] Toxicity of dEpoB vs. EpoB

[0503] The toxicity of EpoB and dEpoB was compared in normal athymicnude mice on the daily i.p. schedule. EpoB at 0.6 mg/kg, QDX4, i.p. ledto lethality in all eight mice. In contrast, in the group treated withdEpoB 25 mg/kg, QDX5, i.p., zero of six mice died. It was also observedthat the vehicle treated control group showed a steady increase in bodyweight and the dEpoB treated mice maintained approximately the sameaverage body weight, whereas the EpoB treated group showed steadydecreases in body weight until death. These results indicated a highertoxicity for both EpoB and dEpoB than in tumor bearing nude mice whenthe treatment schedule was Q2Dx5, i.p. (see Tables 1C and 1D). In thepreliminary studies, for the non-tumor bearing nude mice receiving EpoB0.6 mg/kg or dEpoB 25 mg/kg, QDx4, i.p., there were no apparent changesin hematological cell counts or blood chemistry parameters except for a⁴³% ⁰ decrease in lymphocytes. Similar leukopenia was found withpaclitaxel. Some obstructive fecal mass in the large intestine was notedfollowing Epo treatments in the preliminary study. No gross pathologicalabnormalities were observed in other organs. TABLE 1B Toxicity ofEpothilone B, and Desoxyepothilone B in normal nude mice. Dose scheduleand route of Number of Group administration Number of mice mice diedControl 4 0 Epothilone B 0.6 mg/kg, QD × 4, 8   8* Desoxyepothilone Bi.p. 6 0  25 mg/kg, QD × 4, i.p.

[0504] Comparison of Different Routes of Administration

[0505] Nude mice bearing human ovarian adenocarcinoma, SK-OV3, and humanmammary adenocarcinoma, MX-1, were treated with dEpoB, both i.p. (DMSOas solvent) and i.v. (cremophor and EtOH, 1:1), with Taxol®, i.p. andi.v. (both with clinical samples in cremophor and EtOH as specified bythe manufacturer), and with EpoB, i.v. (used cremophor and EtOH, 1:1).As shown in Table 6, for Q2Dx5 schedule, dEpoB, i.p. (35 mg/kg) andTaxol® i.v. (15 mg/kg) both yield potent therapeutic effects againstMX-1 with tumor-size on day 19, treated/control=0.02 and 0.01,respectively (see Table 1F). For the ovarian tumor a lesser therapeuticeffect was seen, tumor size on day 21, treated/control=0.28 for bothdrugs (see Table 1G). For EpoB i.v., at 0.6 mg/kg there was lesstherapeutic effect and more toxicity than for dEpoB and Taxol®. Incontrast, dEpoB, i.v. (15 mg/kg) and Taxol®, i.p. (5 mg/kg) showed moretoxicity and less therapeutic effect against both tumors. Thus, dEpoBshowed the best results when given i.p. and Taxol® gave better resultswhen given i.v. in cremophor and EtOH.

[0506] In Vitro Effect against Various Tumor Sublines

[0507] Further susceptibility evaluations were conducted for epothilonesA and B and desoxyepothilones A and B in four additional tumor celllines and four of their drug resistant sublines (Table 1C). Hamster lungtumor cells DC-3F/ADX, that were selected 13,000-fold resistant to AD,were found to be 328-fold resistant to paclitaxel and 124-fold resistantto DX when compared with the parent cell line (DC-3F). In contrast,epothilones A and B and desoxyepothilone A showed only 3.9˜28-foldresistance, and epothilones A and B and desoxyepothilone B showed nocross-resistance (0.9-fold resistance).

[0508] Murine leukemic P388/Adr cells that were selected 482-foldresistant to DX, were found to be 111-fold resistant to paclitaxel.However, epothilones A and B showed less than 6-fold resistance, and fordesoxyepothilone A and B there was no cross-resistance (<0.6-foldresistance).

[0509] Human neuroblastoma cells, SK-N—F1, that were selected as 18-foldresistant to DX, were found to be 80-fold resistant to paclitaxel. Bycontrast, epothilone B was 25-fold resistant, while the resistance ofepothilone A and desoxyepothilones A and B was only between 1.9 and 3.1.

[0510] Human mammary carcinoma cells, MCF-7/Adr, that were selected3.8-fold resistant to DX, were found to be 46-fold resistant topaclitaxel. In contrast, compounds epothilones A and B anddesoxyepothilone B was 3.1˜5.4-fold resistant, and dEpoB showed only2.4-fold resistant. Overall, dEpoB was the least cross-resistant amongepothilones and desoxyepothilones in various drug-resistant tumorsublines. By contrast, paclitaxel suffers from marked cross-resistancein tumor cells that were selected to be resistant to VBL, DX or AD. Inthree out of five cell lines studied, cross-resistance to paclitaxel waseven greater than that of the selecting agents.

[0511] Therapeutic Effects Against MX-1 Xenografts

[0512] Therapeutic effects of compounds epothilone A anddesoxyepothilone B, paclitaxel, VBL and CPT were evaluated in athymicnude mice bearing human mammary adenocarcinoma MX-1 xenografts (Table1D). Desoxyepothilone B at a 15 mg/kg dose i.p. on days 7, 9, 11, 13 and15 produced a 50˜60% tumor volume reduction when compared to the controlgroup. A higher dose of drug, 25 mg/kg, produced as much as 96% averagetumor volume reduction measured two days after the last treatment (i.e.,on day 17). These effects were achieved with no lethality nor bodyweight reduction. Furthermore, with a 25 mg/kg dose, one out of six micewas tumor-free on day 35 after tumor implantation (i.e. on day 35). Incontrast, after treatment with EpoB (0.3 mg/kg and 0.6 mg/kg, i.p., ondays 7, 9, 11, 13 and 15), the average body weight decreased over 1 gand 2 g, respectively. In the case of 0.6 mg/kg treatment, three out ofseven mice died of toxicity. Despite the apparent toxicity at thesedoses, EpoB appeared to have only marginal therapeutic effect, as only¹⁶% to ²⁶% tumor volume reduction was observed (Table 1D). The parallelexperiments for paclitaxel led to a lower therapeutic effect. In animalstreated with paclitaxel, 5 mg/kg, there was 55% reduction in tumorvolume and no decrease in average body weight. At a dose of 10 mg/kg,paclitaxel showed a ⁸⁹% tumor reduction, however, four out of seven micedied of toxicity. For DX (2˜3 mg/kg) and CPT (1.5˜3 mg/kg) i.e., nearthe maximal tolerated doses, inferior results were obtained whencompared with dEpoB. Thus, dEpoB even at non-toxic dose had the besttherapeutic effect among the five compounds studied underthe sameexperimental conditions.

[0513] In a separate experiment, MX-1 xenograft-bearing mice weretreated with dEpoB, 35 mg/kg, Q2Dx5, i.p. beginning on day 8 after tumorimplantation (FIG. 60). On day 16, two out of ten mice had no detectabletumor. These ten mice were further treated with dEpo B, 40 mg/kg, Q2Dx5beginning on day 18. At the end of treatment on day 26, five out of tenmice had no detectable tumor, and three remained tumor-free on day 60.There was body weight reduction during treatments but no lethalityoccurred. In a parallel experiment, ten mice were treated withpaclitaxel, 5 mg/kg, Q2Dx5, i.p. from day 8 to day 16, followed by asecond cycle of treatment in the same manner from day 18 to day 26. Thetumor sizes were reduced but continued to grow during treatment and byday 24, the average tumor size was 2285±597 mm³ (n=10). In a parallelexperiment, DX was given 2 mg/kg, Q2Dx5, i.p. (FIG. 60), and reducedtherapeutic effect was seen compared to dEpoB or paclitaxel. No dataafter day 18 is shown because the tumor burden in the control group wasexcessive and the mice in this group were sacrificed.

[0514] Therapeutic Effects Against MCF-7/Adr Xenografts

[0515] The therapeutic effects of dEpoB, Taxol®, DX and CPT were alsoevaluated in nude mice bearing xenografts of human mammaryadenocarcinoma resistant to DX (MCF-7/Adr) (Table 1E). As indicatedearlier in Table 1B for the cytotoxicity results in vitro, MCF-7/Adrcells selected to be 3.8-fold resistant to DX were found to be 46-foldresistant to paclitaxel, and only 2.4-fold resistant to dEpoB. For invivo studies, each drug was given Q2Dx5, i.p. beginning on day 8 aftertumor implantation. Paclitaxel 12 mg/kg and DX 3 mg/kg were highly toxicto the nude mice with 3/7 and 3/6 lethality, respectively. CPT 3 mg/kgled to moderate toxicity without lethality, and dEpoB 35 mg/kg showednegligible toxicity as shown by minimal body weight changes (Table 1E).CPT at 3 mg/kg reduced 57% of tumor size on day 17 (p<0.05 when comparedwith control group). Desoxyepothilone B at 35 mg/kg significantlysuppressed tumor size by 66-73% when compared with the control group(p<0.005-0.05), without complete tumor regression. In contrast,paclitaxel 5 mg/kg, and DX 2 mg/kg, produced slight growth suppressionof this drug-resistant tumor which was not significantly different fromthe control group (see Table 1D). Thus, dEpoB stands out as the superiordrug among the four tested against this drug-resistant tumor. TABLE 1CComparison of in vitro growth inhibition potency of epothilonederivatives against various parent and drug resistant tumor cell lines.DC- P388/ SK-N- SK-N- MCF-7/ Compound DC-3F 3F/ADX P388/0 Adr As FlMCF-7 Adr IC₅₀ (μM)* Epo A 0.0037  0.053 0.0018 0.0010 0.012 0.0230.0030 0.0094 (14.5x)^(#) (5.3x)^(#) (1.9x)^(#) (3.1x)^(#) Epo B 0.0006 0.017 0.00029 0.0016 0.004 0.010 0.0005 0.0027 (28x) (5.5x) (25x)(5.4x) dEpo A 0.011  0.042 0.0213 0.0125 0.073 0.223 0.032 0.144 (3.9x)(0.59x) (3.1x) (4.5x) dEpo B 0.00097  0.00091 0.0068 0.0042 0.021 0.0460.0029 0.0071 (0.9x) (0.62x) (2.2x) (2.4x) Taxol ® 0.095 32.0 0.00290.326 0.0016 0.130 0.0033 0.150 (338x) (111x) (80x) (46x) Actinomycin0.00044  0.572 0.00015 0.0012 0.00085 0.0119 0.00068 0.00167 D (13000x)(8x) (14x) (2.5x) 0.018  2.236 0.0055 2.65 0.077 1.42 0.057 0.216Adriamycin (124x) (482x) (18.4x) (3.8x) #antagonism, low-dose risk,reception-ligand binding and enzyme kinetics, 1987, IBM-PC software andmanual. Biosoft, Cambridge, U.K.).

[0516] TABLE 1D Therapeutic effect of desoxyopothilone B, epothilone B,Taxol ®, vinblastine and camptothecin in nude mice bearing human MX-1zenograft. Dose Average Body Weight Change (g) Average Tumor Size (T/C)Toxicity Drug (mg/kg) Day 7 11 13 15 17 Day 11 13 15 17 Death N Control27.2 +0.8 +1.1 +1.9 +0.6 1.00 1.00 1.00 1.00 0/8 8 dEpo B 15 27.1 +0.8+1.1 +1.6 +1.5 0.65 0.46 0.49** 0.41** 0/6 6 25^(#) 27.0 +0.4 +0.7 +1.0+0.7 0.38* 0.11** 0.05*** 0.04**** 0/6 6 Epo B 0.3 26.9 +0.5 +0.4 −0.3−1.2 1.00 0.71 0.71 0.84 0/7 7 (0.6^(α) 27.4 −0.3 −1.3 −2.1 −2.1 1.080.73 0.81 0.74 3/7)^(##) 7 Taxol ® 5 26.9 −0.1 +0.4 +1.1 +1.2 0.54 0.460.40* 0.45** 0/7 7 10^(Δ) 27.6 −2.7 −1.1 −0.3 +2.2 0.43 0.37 0.12 0.114/7^(##) 7 Vinblastine 0.2 25.7 +0.6 +1.4 +2.3 +2.9 0.65 0.54 0.56 0.880/7 7 (0.4^(β) 26.4 +0.8 +0.5 +1.9 +2.1 0.80 0.56 0.83 0.88 1/7)^(##) 7Campothecin 1.5 27.4 −0.9 −0.7 −0.4 +1.0 0.61 0.45* 0.32* 0.36** 0/7 7

[0517] TABLE 1E Therapeutic effects of desoxyopothilone B, epothilone B,taxol, adriamycin and camptothecin in nude mice bearing MDR humanMCF-7/Adr tumor. Dose Average Body Weight Change (g) Average Tumor Size(T/C) Toxicity Drug (mg/kg) Day 8 11 13 15 17 Day 11 13 15 17 Death NControl 0 25.0 +2.0 +2.6 +3.1 +3.7 1.00 1.00 1.00 1.00 0/8 8 dEpo B 3525.0 0.3 +0.7 +0.6 +0.8 0.31** 0.27*** 0.30*** 0.34* 0/8 8 Taxol ® 625.3 +1.7 +1.8 +0.8 +0.9 0.57 0.66 0.85 0.90 0/7 7  (12 24.5 −0.7 −1.3−2.4 0 0.50 0.51 0.32 0.40 3/7 7)^(#) Adriamycin 2 25.6 +0.2 −0.4 −0.6−0.4 0.70 0.68 0.84 0.78 0/8 8 (3 24.6 +0.5 −1.3 −3.2 −1.6 0.66 0.830.57 0.53 3/6 6)^(#) Campothecin 1.5 24.4 +1.1 +0.9 +1.7 +1.4 1.08 0.720.61 0.72 0/8 8 (3 24.5 −0.6 −0.4 −0.8 −0.9 0.95 0.76 0.61 0.43* 0/6 6

[0518] TABLE 1F Therapeutic effects of desoxyopothilone B, Epo B andTaxol ® in nude mice bearing MX-1 tumors using different vehicles anddifferent routes of administration.^(a) Dose Average Body Weight Change(g) Average Tumor Size (T/C) Tumor Toxicity Drug/Route (mg/kg) Day 9 1315 17 19 Day 13 15 17 19 Disapp. Death Control 0 26.4 −0.2 −0.4 +0.2+0.8 1.00 1.00 1.00 1.00 0/6 0/6 dEpo B/i.p. 35 27.8 −1.7 −2.1 −2.1 −2.40.35 0.14 0.04 0.02 3/6 0/6 dEpo B/i.v. 15 27.0 0 −0.6 −1.1 −2.6 0.470.30 0.10 0.04 0/6 4/6^(b) Epo B/i.p. 0.6 27.0 −0.9 −0.5 −3.3 −3.4 0.670.63 0.61 0.51 0/6 0/6 Taxol ®/i.p. 5 27.4 −1.1 −2.0 −1.0 −0.2 0.59 0.720.59 0.55 0/6 0/6 Taxol ®/i.v. 15 27.2 −0.6 −0.8 −0.8 −0.9 0.36 0.130.04 0.01 2/6 0/6

[0519] TABLE 1G Therapeutic effects of desoxyopothilone B, Epo B andTaxol ® in nude mice breaking SK-OV-3 tumors using different vehiclesand different routes of administration.^(a) Dose Average Body WeightChange (g) Average Tumor Size (T/C) Tumor Toxicity Drug/Route (mg/kg)Day 9 13 15 17 19 Day 13 15 17 19 Disapp. Death Control 0 26.4 −0.2 −0.4+0.2 +0.8 1.00 1.00 1.00 1.00 0/6 0/6 dEpo B/i.p. 35 27.8 −1.7 −2.1 −2.1−2.4 0.57 0.33 0.35 0.28 0/6 0/6 dEpo B/i.v. 15 27.0 0 −0.6 −1.1 −2.60.86 0.56 0.50 0.44 0/6 4/6^(b) Epo B/i.p. 0.6 27.0 −0.9 −0.5 −3.3 −3.40.75 0.69 0.88 0.77 0/6 0/6 Taxol ®/i.p. 5 27.4 −1.1 −2.0 −1.0 −0.2 0.690.60 0.49 0.40 0/6 0/6 Taxol ®/i.v. 15 27.2 −0.6 −0.8 −0.8 −0.9 0.970.67 0.42 0.28 0/6 0/6

[0520] Discussion

[0521] Two classes of naturally occurring compounds, epothilones andpaclitaxel, which are apparently structurally dissimilar, show similarmodes of action in stabilizing microtubule assemblies. Thesesimilarities include binding tubulin, substitution for paclitaxel inmaintaining paclitaxel-dependent cell growth in a resistant cell line,and similar morphologic changes as determined by electron microscopicexamination of the drug-microtubule complex. There are differences,however, between the two classes of compounds. These differences aremost prominently exhibited by the lack of cross-resistance incytotoxicity between the epothilones and paclitaxel even inCCRF-CEM/Taxol® cells (Table 1A). Furthermore, in CCRF/CEM/VBL₁₀₀, thecells were 527-fold resistant to vinblastine and 1971-fold resistance topaclitaxel, but were only 6.1-fold resistant to EpoB and 1.8-foldresistant to dEpoB (Table 1A). In DC-3F/ADX cells, there was 13,000-foldresistance to actinomycin D and 338-fold resistance to paclitaxel.However, these cells were only 28-fold resistance to EpoB and had noresistance to dEpoB (i.e., 0.9-fold resistance or collateralsensitivity) (Table 1B). Paclitaxel showed a higher degree ofcross-resistance in these cell lines than other MDR-drugs such asdoxorubicin, actinomycin D, vinblastine or etoposide. In some cases thedegrees of resistance to paclitaxel were even greater than those of theresistance-selecting agent (e.g., CCRF-CEM/VBL₁₀₀ in Table 1A, andSK-N-FI and MCF/7-Adr in Table 1B). In contrast, among al I compoundstested, dEpoB showed the least cross-resistance in severaldrug-resistant cell lines (e.g. DC-3F/Adr) and even showed slightcollateral sensitivity (e.g. DC-3F/ADX and P388/Adr in Table 1B).Parallel cancer chemotherapeutic studies for EpoB, dEpoB, Taxol® andother drugs were performed under the same experimental conditions (i.e.,treatment schedule, Q2D; solvent vehicle, DMSO; and route ofadministration, i.p.) in animals.

[0522] The i.p. route of other formulations for administration of dEpo Bis far better tolerated than the i.v. method. Even though EpoB is themost potent, it is by no means the best candidate for cancer therapy interms of therapeutic index (i.e. the therapeutic efficacy at tolerabledosage, or the ratio of toxic dose vs the therapeutic dose).Desoxyepothilone B, lacking the epoxide functionality, exhibited farsuperior therapeutic results in vivo as compared to the more potentEpoB. Similarly, the present therapeutic results for dEpoB in MX-1xenografts were far better than those for EpoB, paclitaxel, doxorubicin,vinblastine or camptothecin. In addition, the effects of dEpo B onMCF-7/Adr xenografts were significantly better than those forpaclitaxel, doxorubicin and camptothecin. In view of the finding thatthe epothilones have little or no cross-resistance against MDR tumorcells in vitro, the special therapeutic advantage of such compoundsmight be in their use against MDR-resistant tumors.

[0523] Novel Aldol Condensation with 2-Methyl-4-pentenal: Application toPreparation of Epothilone B and Desoxyepothilone B

[0524] Stereoselectivity poses a potential hindrance to enhancing accessto multicomponent libraries. Nicolaou, K. C., et al., J. Am. Chem. Soc.1997, 119, 7960; Nicolaou, K. C., et al., J. Am. Chem. Soc. 1997, 119,7974. Nicolaou, K. C., et al., Angew. Chem. Int. Ed. Engl. 1997, 36,2097. However, stereoselectivity holds the attraction that it allows foraccumulation of substantial quantities of fully synthetic keyepothilones of correct configuration. Comparable harvesting of neededamounts of material through the stereo-random olefin metathesis route(Yang, Z., Y., et al., Angew. Chem. Int. Ed. Engl. 1997,36, 166;Nicolaou, K. C., et al., Angew. Chem. Int. Ed. Engl. 1997, 36, 525;Nicolaou, K. C., et al., Nature 1997, 387, 268; Nicolaou, K. C., et al.,J. Am. Chem. Soc. 1997, 119, 7960; Nicolaou, K. C. et al., J. Am. Chem.Soc. 1997, 119, 7974; Nicolaou, K. C., et al., Angew. Chem. Int. Ed.Engl. 1996, 35, 2399. Schinzer, D., et al., Angew. Chem. Int. Ed. Engl.1997, 36, 523; Meng, D., et al., J. Am. Chem. Soc. 1997, 119, 2733)would be virtually prohibitive. Biological studies in xenograft miceprovided herein identified some significant toxicity problems with thehighly potent epothilone B. Remarkably, in vivo studies in theintraperitoneal mode of injection demonstrate that the less potent12,13-deoxyepothilone B is well tolerated and is virtually curativeagainst a variety of xenograft tumors. Chou, T. -C., et al., Proc. Nat'lAcad. Sci. USA 1998, 0000. Desoxyepothilone B has clinical advantagesrelative to paclitaxel, particularly as regards vulnerability to thephenomenon of multiple drug resistance. The preparative route disclosedherein retains the advantages of high stereoselectivity throughout, andprovides an improved approach to the previously difficult C1-C11 domain.FIG. 53 provides a global overview of the problem.

[0525] The route disclosed herein is based on four findings. The firstis the ease of formation and the synthetic utility of the Z-lithiumenolate (48A) readily produced from 59 as shown in FIG. 61(A). In thiseasily obtained construct, the critical enolate of the ethyl ketone isfashioned in the context of a putative, 5-diketoester ensemble embracingcarbons 1-6 of the target (cf. structure 47). The advantages of thisdirect approach for synthetic economy are apparent.

[0526] The second and most surprising finding undergirding thissynthesis is that the sense of addition of enolate 48A to readilyavailable S-aldehyde 58 provides the desired C₇-C₈ anti relationshipwith good diastereofacial selectivity in conjunction with the expectedC₆-C₇ syn relationship (by Ik-addition). C. H. Heathcock, in AsymmetricSynth. J. D. Morrison, ed., Academic Press, New York, 1984, Vol.3,p.111-212; see compound 49 and its stereoisomer. The stereochemistry ofthe minor diastereomer was presumed to be C₇-C₈ syn, but was notrigorously proven. This was based on precedent (Mori, I., et al., J.Org. Chem., 1990, 55, 1114), assuming that this isomer arises fromfacial selectivity in the aldol reaction, not as a consequence of theE-enolate of 48A. Those assignments also rely to varying extents on theHeathcock precedent. The 5.5:1 outcome for the diastereofacialselectivity of this aldol reaction is counter to expectations arisingfrom the traditional models first advanced by Cram and Felkin. (Fortransition state models in diastereomeric carbonyl addition reactionssee: D. J. Cram, F. A. Elhafez, J. Am. Chem. Soc. 1952, 74, 5828; D. J.Cram, K. R. Kopecky, J. Am. Chem. Soc. 1959, 81, 2748; J. W. Cornforth,R. H., et al., J. Am. Chem. Soc. 1959, 81, 112; G. J. Karabatsos, J. Am.Chem. Soc. 1967, 89, 1367; M. Cherest, H. Felkin, N. Prudent,Tetrahedron Lett. 1968, 2199; N. T. Ahn, O. Eisenstein, Nouv. J. Chem.1977, 1, 61; A. S. Cieplak, J. Am. Chem. Soc. 1981, 103, 4540; E. P.Lodge, C. H. Heathcock J. Am. Chem. Soc. 1987, 109, 2819.) Theseextensively invoked formulations, which differ widely in theirunderlying conformational assumptions and stereochemical treatments,usually converge in terms of their predicted outcome.

[0527] The high anti:syn diastereofacial ratio arises from a peculiarcharacteristic of aldehyde 58 and likely reflects the relationship ofits vinyl and formyl groups. It is not, apparently the result of a grossproperty of enolate 48A. Indeed, the same enolate, with the benchmarkaldehyde phenylpropanal 60a, performs in the expected fashion (C. H.Heathcock in Asymmetric Synth. J. D. Morrison, ed., Academic Press, NewYork, 1984, Vol. 3, p.111-212), yielding an 11:1 ratio of 61a:62a.Furthermore, with aldehyde 60b, the dihydro version of 58, the C7 to C8anti:syn (61b:62b) ratio drops to 1:1.3. Moreover, when the distancebetween the vinyl and formyl groups is extended, as in 60c, selectivityis also compromised. By contrast the phenyl and dimethylallyl analogs of58 (60d and 60e) bearing the same relationship of unsaturated groups asin 58, exhibit good anti-antiselectivity (see products 61d and 61e aswell as 62d and 62e). Also, aldehyde 60f, a substrate known for itstendency to favor the anti-diastereofacial product (M. T. Reetz, Angew.Chem. Int. Ed. Engl. 1984, 23, 556) on the basis of presumed chelationcontrol, performs normally with enolate 48A affording a 1:4 ratio of 61f:62f.

[0528] With respect to the impact of the strong anti:syn diastereofacialselectivity in the aldol reaction of 58 and 48A on the overallefficiency of the synthesis, the rather favorable result in establishingthe C₇-C₈ bond opened the possibility that the C₁-C₇ fragment could beentered into the synthesis as an achiral block. Accordingly, it would benecessary to gain control over the eventual stereochemistry at C3. Thissubgoal was to be accomplished by the implementation of any asymmetric,reagent controlled Noyori reduction (vide infra). Noyori, R., et al., J.Am. Chem. Soc. 1987, 109, 5856; Taber, D. F., Silverbert, L.,Tetrahedron Lett. 1991, 32, 4227; Ager, D. J., Laneman, S., TetrahedronAsymmetry 1997, 8, 3327.

[0529] The third critical element was the finding that the key B-alkylSuzuki merger which controls the geometry of the trisubstituted doublebond can be conducted successfully even on the elaborate 51, obtainedfrom 49. The cognate substrate for the Suzuki reaction was thepreviously described vinyl iodide, 51 (Su, D. -S., et al., Angew. Chem.Int. Ed. Engl. 1997, 36, 757). The remarkable coupling step, affordedthe Z-olefin 52A and thence, 52 after removal of the C15 silylprotecting group (FIG. 61(B)). The β,δ-disketo ester array in 52responded well to asymmetric catalytic reduction under modified Noyoriconditions (Taber, D., Silverbert, L. J., Tetrahedron Lett. 1991, 32,4227) to give the diol 53 (88%, >95:5). Strict regiochemical anddiastereofacial control in the Noyori reduction was very dependent onthe amount of acid present in the reaction. Without acid, the rate ofreduction dropped off as well as the selectivity in the reduction.Further, the carbonyl at C-5 was never reduced under these conditionsbut was absolutely necessary for the reduction of the C-3 carbonyl. WhenC-5 was in the alcohol oxidation state, no reduction was seen. Theconversion of 53 to desoxyepothilone B and thus epothilone B wasaccomplished by methodologies set forth herein. Balog, A., et al.,Angew. Chem. Int. Ed. Engl. 1996, 35, 2801; Su, D. -S., et al., Angew.Chem. Int. Ed. Engl. 1997, 36, 757; Meng, D., et al., J. Am. Chem Soc.1997, 119, 10073.

[0530] Biological Results

[0531] In the tables which follow, model system I is desoxyepothilone.Model system 2 has the structure:

[0532] wherein R′ and R″ are H.

[0533] Model system 3 has the structure:

[0534] As shown in Table 2A, CCRF-CEM is the parent cell line.CCRF-CEM/VBL (MDR cell line) is 1143-fold resistant to Taxol®.CCRF-CEM/VM (Topo II mutated cell line) only 1.3-fold resistant toTaxol®.

[0535] In terms of relative potency, synthetic Epothilone is roughly thesame as natural Epothilone A. For CCRF-CEM cells, the ordering is:

[0536] Taxol®≈Epothilone A>Desoxy Epothilone A>>Triol Analog >>ModelSystem I

[0537] For CCRF-CEM/VBL, the relative potency ordering is:

[0538] Desoxy Epothilone A≧Epothilone A>>Taxol®>Triol Analog >ModelSystem I

[0539] For CCRF-CEM/VM, the relative potency ordering is:

[0540] Taxol®≈Epothilone A>Desoxy Epothilone A>>Model System I>TriolAnalog

[0541] It is concluded that CCRF-CEMNM cells are collaterally sensitiveto certain epothilone compounds. TABLE 2 Relative Efficacy of EpothiloneCompound Against Human Leukemic CCRF-CEM Cell Growth and AgainstCCRF-CEM MDR Sublines Resistant to Taxol ® or Etoposide IC₅₀ in μMCCRF-CEM/ CCRF-CEM/ COMPOUND CCRF-CEM VLB VM-1 EPOTHILONE A NATURAL0.0035 0.0272 0.0034 EPOTHILONE A 0.0029 0.0203 0.0034 SYNTHETIC MODELSYSTEM I [3] 271.7 22.38 11.59 TRIOL ANALOG [2] 14.23 6.28 43.93 DESOXYEPOTHILONE [1] 0.002 0.012 0.013 Taxol ® 0.0023 2.63 0.0030 VINBLASTINE0.00068 0.4652 0.00068 VP-16 (ETOPOSIDE) 0.2209 7.388 34.51

[0542] TABLE 2A Relative Potency of Epothilone Compounds Against HumanLeukemic CCRF Sublines CCRF-CEM/VBL CCRF-CEM/VM₁ (MDR Cell Line) (TopoII gene mutated cell line) CRF-CEM (Taxol ® Resistant)-(1143 (Taxol ®Sensitive) (Parent Cell Line) fold) (Vinblastine Resistant) (VP-16resistant) IC₅₀ [IC₅₀ IC₅₀ [IC₅₀ IC₅₀ [IC₅₀ (μM) relative to (μM)relative to (μM) relative to COMPOUND (A) Epothilone A] (B) Epothilone A(B)/(A)] (C) Epothilone A (C)/(A)] Taxol ®  0.0023 [0.72]  2.63 [109.6](1143)^(a)  0.0030 [0.88] (1.30)^(a) MODEL 271.7 [84906] 22.38 [932.5](0.082)^(b) 11.59 [3409] (0.043)^(b) SYSTEM I TRIAL ANALOG  14.23 [4447] 6.28 [261.7] (0.44)^(b) 43.93 [12920] (3.09)^(a) DESOXYEPOTHILONE 0.022 [6.9]  0.012 [0.5] (0.55)^(b)  0.013 [3.82] (0.59)^(b) AEPOTHILONE A  0.0032 [1]  0.024 [1] (7.5)^(a)  0.0034 [1] (1.06)^(a)

[0543] TABLE 3 Relative Efficacy of Epothilone Compounds Against TheDC-3F Hamster Lung Cell Growth and Against DC-3F MDR Sublines ResistantActinomylin D IC₅₀ in μM COMPOUNDS DC-3F DC-3F/ADII DC-3F/ADX EPOTHILONEA 0.00368 0.01241 0.0533 NATURAL EPOTHILONE A 0.00354 0.0132 0.070SYNTHETIC MODEL SYSTEM I [3] 9.52 3.004 0.972 TRIOL ANALOG [2] 10.324.60 4.814 DESOXY EPOTHILONE 0.01061 0.0198 0.042 [1] Taxol^(®) 0.094693.205 31.98 VINBLASTINE 0.00265 0.0789 1.074 VP-16 (Etoposide) 0.033860.632 12.06 ACTINOMYCIN-D 0.000058 0.0082 0.486 (0.05816 nm)

[0544] Concerning Table 3, experiments were carried out using the celllines DC-3F (parent hamster lung cells), DC-3F/ADII (moderatemultidrug-resistant (MDR) cells) and DC-3F/ADX (very strong MDR cells).

[0545] The relative potency of the compounds are as follows:

[0546] DC-3F: Actinomycin D>Vinblastine >Epothilone A (0.0036 μM)>Desoxyepothilone>VP-16>Taxol® (0.09 μM)>Model system I and triol analog

[0547] DC-3F/ADX: Desoxyepothilone≧Epothilone A (0.06 μM)>ActinomycinD>Model system I>Vinblastine>triol analog>viablastine>Taxol® (32.0 μM)

[0548] DC-3F/ADX cells (8379-fold resistant to actinomycin D) are>338fold (ca. 8379 fold) resistant to Taxol®, VP-16, Vinblastine andActinomycin D but<20 fold resistant to epothilone compounds.

[0549] In general, these results are similar to those for CCRF-CEMcells. TABLE 4 Three Drug Combination Analysis (Based on the MutuallyExclusive Assumption - Classical Isobologram Method) Dm CombinationIndex* Values at: (IC₅₀) Parameters Drug ED50 ED75 ED90 ED95 (μM) m r A−00061 1.71561 .98327 B −00109 2.14723 .98845 C −00061 1.76186 .9919 A +B 1.51545 1.38631 1.27199 1.20162 −00146 2.41547 .97168 B + C 1.432431.33032 1.23834 1.18091 .00138 2.35755 .95695 A + C .74395 .68314 .62734.59204 .00045 2.0098 .96232 A + B + C 1.37365 1.32001 1.27285 1.24412.00122 2.11202 .93639 VBL → microtubule depolymerization Taxol ® →microtubule polymerization Epo-B → microtubule polymerization EpothiloneB and Taxol ® have a similar mechanism of action (polymerization) butEpothilone B synergizes VBL whereas Taxol ® antagonizes VBL. Taxol ® +VBL → Antagonism EpoB + Taxol ® → Antagonism EpoB + VBL → SynergismEpoB + Taxol ® + VBL → Antagonism

[0550] TABLE 5 Relative cytotoxicity of epothilone compounds in vitro.IC₅₀ in μM Compounds CCRF-CEM CCRF-CEM/VLB CCRF-CEM/VM-1 VINBLASTINE ***0.0008  0.44 (52.7X)^(§)  0.00049 (0.7X) 0.0006 (0.00063 ±  0.221 (0.332 0.00039 (0.00041 ± 0.0005 0.00008)  0.336 ± 0.063  0.00036 0.00004)VP-16 0.259  6.02 (35.3X) 35.05 (117.4X) 0.323 (0.293 ±  9.20 (10.33 ±42.24 (34.39 ± 0.296 0.019) 15.76 2.87) 25.89 4.73) Taxol ® *** 0.0021 4.14  0.0066 #17 * 0.090 0.254 #18 1157.6 >>1 #19 0.959 >>1 #20 * 0.0300.049 #21 — — #22 * 0.098 0.146 #23 — — #24 *** 0.0078 0.053 #25 * 0.021 0.077 #26 *  0.055 0.197 #27 **** 0.0010 0.0072 Epothilone A ***0.0021 0.015 (Syn) Epothilone B **** 0.00042 0.0017 (Syn)

[0551] TABLE 6 Relative potency of epothilone compounds in vitro. IC₅₀in μM Compounds CCRF-CEM CCRF-CEM/VBL CCRF-CEM/VM-1 Desoxy Epo. A 1 *0.022 0.012 0.013 2 14.23 6.28 43.93 3 271.7 22.38 11.59 4 2.119 43.012.76 5 >20 35.19 98.04 Trans- A 6 0.052 0.035 0.111 7 7.36 9.82 9.65Syn-Epo.-B 8 **** 0.00082 0.0029 0.0044 Natural B 9 **** 0.00044 0.00260.0018 Desoxy Epo. B 10 *** 0.0095 0.017 0.014 Trans. Epo. B 11 * 0.0900.262 0.094 12 0.794 >5 >5 13 11.53 5.63 14.46 8-desmethyl desoxy-Epo 145.42 5.75 6.29 8-desmethyl Mix-cis Epo 15 0.96 5.95 2.55 8-desmethylβ-Epo 15 0.439 2.47 0.764 8-demethyl α-Epo 16 7.47 16.48 0.976EPOTHILONE A *** 0.0024 (0.0027 ± 0.0211 (0.020 ± 0.006 (0.00613 ±(Natural) 0.0031 0.0003) 0.0189 0.001) {close oversize brace} 0.0001)0.00625 (7.4X) (2.3X) EPOTHILONE B **** 0.00017 0.0017 (7.0X) 0.00077(Natural) EPOTHILONE B (Synthetic) 0.00055 0.0031 (0.00213 ± 0.0018(0.00126 ± EPOTHILONE B (0.00035 ± 0.00055) 0.0003) (Synthetic, larger0.0003) quantity 0.0021 (6.1X) 0.0012 (3.6X) synthesis) 0.00033 (25.9mg)

[0552] TABLE 7 Relative cytotoxicity of epothilone compounds in vitro.IC₅₀ CEM CEM/VBL epothilone A 0.0029 μM 0.0203 μM desoxyepothilone 0.0220.012  2 14.2 6.28  3 271.7 22.4  4 2.1 43.8  5 >20 35.2  6 0.052 0.035 7 7.4 9.8 synthetic epothilone B 0.00082 0.00293 natural epothilone B0.00044 0.00263 desoxyepothilone B 0.0095 0.0169 11 0.090 0.262 120.794 >5 13 11.53 5.63 14 5.42 5.75 15 0.439 2.47 16 7.47 16.48 17 0.0900.254 18 1157.6 >>1 19 0.959 >>1 20 0.030 0.049 21 Not Available — 220.098 0.146 23 Not Available — 24 0.0078 0.053 25 0.0212 0.077 26 0.05450.197 27 0.0010 0.0072

[0553] TABLE 8 Chemotherapeutic Effect of Epothilone B, Taxol ® &Vinblastine in CB-17 Scid Mice Bearing Human CCRF-CEM and CCRF-CEM/VBLXenograft¹ Average weight change Average tumor volume Day Day Day DayDay Day Day Day Day Tumor Drug² Dose 0 7 12 17 22 7 12 17 22 CCRF-CEM 024.4 +0.2 +0.4 +0.1 +0.5 1.0³ 1.00 1.00 1.00 Epo B 0.7⁴ 24.7 −0.1 −0.7−1.4 +0.3 1.0 0.53 0.48 0.46 1.0⁵ 25.0 +0.1 −1.5 −2.4 +0.1 1.0 0.46 0.350.43 Taxol ® 2.0 25.1 −0.1 −1.1 −1.5 −0.3 1.0 0.39 0.29 0.28 4.0 25.1−0.2 −1.7 −1.9 −0.3 1.0 0.37 0.23 0.19 VBL 0.2 25.9 +0.2 −0.8 −1.5 −0.31.0 0.45 0.25 0.29 0.4 25.0 −0.1 −1.4 −1.8 −0.7 1.0 0.31 0.27 0.30CCRF-CEM/VBL 0 26.3 −0.3 +0.1 −0.3 +0.4 1.0 1.00 1.00 1.00 Epo B 0.725.8 +0.1 −0.7 −1.0 −0.2 1.0 0.32 0.40 0.33 1.0⁶ 26.0 −0.2 −1.3 −2.1−0.5 1.0 0.41 0.27 0.31 Taxol ® 2.0 26.1 0 −0.9 −1.5 −0.1 1.0 0.60 0.580.70 4.0 26.0 0 −1.4 −1.6 −0.9 1.0 0.79 0.55 0.41 VBL 0.2 25.9 −0.3 −0.8−1.4 −0.3 1.0 0.86 0.66 0.67 0.4 25.9 0 −1.2 −1.8 −0.5 1.0 1.02 0.570.62

[0554] In summary, epothilones and Taxol® have similar modes of actionby stabilizing polymerization of microtubules. However, epothilones andTaxol® have distinct novel chemical structures.

[0555] MDR cells are 1500-fold more resistant to Taxol® (CCRF-CEM/VBLcells), epothilone A showed only 8-fold resistance and epothilone Bshowed only 5-fold resistance. For CCRF-CEM cells, Epo B is 6-fold morepotent than Epo A and 10-fold more potent than Taxol®. DesoxyepothiloneB and compd #24 are only 3-4-fold less potent than Taxol® and compound#27 is >2-fold more potent than Taxol®. Finally, Taxol® and vinblastineshowed antagonism against CCRF-CEM tumor cells, whereas the combinationof Epo B+vinblastine showed synergism.

[0556] Relative Cytotoxicity of Epothilones against Human Leukemic Cellsin Vitro is in the order as follows:

[0557] CCRF-CEM Leukemic Cells

[0558] EpoB(IC₅₀=0.00035 μM; Rel. Value=1)>VBL(0.00063;1/1.8)>#27(0.0010; 1/2.9)>Taxol® (0.0021; 1/6)>Epo A (0.0027;1/7.7)>#24(0.0078; 1/22.3)>#10 (0.0095; 1/27.1)>#25 (0.021; 1/60)>#1(0.022; 1/62.8)>#20 (0.030; 1/85.7)>#6 (0.052; 1/149)>#26 0.055;1/157)>#17(0.090; 1/257)>VP-16 (0.29; 1/8.29)>#15 (0.44; 1/1257)>#19(0.96; 1/2943)

[0559] CCRF-CEM/VBL MDR Leukemic Cells

[0560] EpoB(0.0021; 1/6* [1]**)>#27(0.0072; 1/20.6)>#1 (0.012;1/34.3)>#10(0.017; 1/48.6)>Epo A (0.020; 1/57.1 [1/9.5])>#6 (0.035)>#20(0.049)>#24 (0.053)>#25 (0.077)>#22 (0.146)>#26 (0.197)>#17 (0.254)>#11(0.262)>VBL (0.332; 1/948.6 [1/158.1])>Taxol® (4.14; 1/11828[1/1971.4])>VP-16 (10.33; 1/29514 [1/4919])

[0561] *Potency in parentheses is relative to Epo B in CCRF-CEM cells.

[0562] **Potency in square brackets is relative to Epo B in CCRF-CEM/VBLMDR cells.

[0563] As shown in Table 9, treatment of MX-1 xenograft-bearing nudemice with desoxyepothilone B (35 mg/kg, 0/10 lethality), Taxol® (5mg/kg, 2/10 lethality; 10 mg/kg, 2/6 lethality) and adriamycin (2 mg/kg,1/10 lethality; 3 mg/kg, 4/6 lethality) every other day, i.p. beginningday 8 for 5 doses resulted in a far better therapeutic effect fordesoxyepothilone B at 35 mg/kg than for Taxol® at 5 mg/kg and adrimycinat 2 mg/kg with tumor volume reduction of 98%, 53% and 28%,respectively. For the desoxyepothilone B-treated group, 3 out of 10 micewere found with tumor non-detectable on day 18. (See FIG. 46)

[0564] Extended treatment with desoxyepothilone B (40 mg/kg, i.p.)beginning day 18 every other day for 5 more doses resulted in 5 out of10 mice with tumor disappearing on day 28 (or day 31). See Table 10. Bycontrast, the extended treatment with Taxol® at 5 mg/kg for five moredoses resulted in continued tumor growth at a moderate rate, and 2 outof 10 mice died of toxicity.

[0565] Toxicity studies with daily i.p. doses of desoxyepothilone B (25mg/kg, a very effective therapeutic dose as indicated in earlierexperiments) for 4 days to six mice resulted in no reduction in averagebody weight. (Table 13; FIG. 47) By contrast, epothilone B (0.6 mg/kg,i.p.) for 4 days to eight mice resulted in 33% reduction in average bodyweight; all eight mice died of toxicity between day 5 and day 7.

[0566] As evident from Table 15, desoxyepothilone B performssignificantly better than Taxol®, vinblastine, adriamycin andcamptothecin against MDR tumor xenografts (human mammary adeoncarcinomaMCF-7/Adr xenografts). This drug-resistant tumor grows very aggressivelyand is refractory to Taxol® and adriamycin at half their lethal doses.Taxol® at 6 mg/kg i.p. Q2Dx5 reduced tumor size only 10% whileadriamycin resulted in only a 22% reduction on day 17. Whereas,desoxyepothilone B at 35 mg/kg reduced tumor size by 66% on day 17 andyet showed no reduction in body weight or apparent toxicity. Even at theLD₅₀ dosage for Taxol® (12 mg/kg) or adriamycin (3 mg/kg),desoxyepothilone B still performed more effectively. By comparison,camptothecin at 1.5 and 3.0 mg/kg reduced tumor size by 28% and 57%,respectively. Overall, in comparison with the four important anticancerdrugs in current use, i.e., Taxol®, adriamycin, vinblastine andcamptothecin, desoxyepothilone B showed superior chemotherapeutic effectagainst MDR xenografts.

[0567] In vivo therapeutic results in nude mice for dEpoB and Taxol® arereported in Tables 19-21. As shown in the Tables, 6 hr i.v. infusion viatail vein provided a good therapeutic profile with remarkably lowtoxicity.

[0568] For mammary MX-1 xenograft (non-MDR), desoxyepothilone B was aseffective as Taxol®. Both drugs were administered by 6 hr i.v. infusionand both achieved full cure.

[0569] For the MDR-mammary MCF-7/Adr xenograft, the therapeutic effectof desoxyepothilone B was far better than Taxol®, although Q2Dx5 did notachieve a cure.

[0570] For CCRF-CEM/Taxol® (57-fold resistant to Taxol® in vitro,in-house developed cell line), Taxol® did not show significanttherapeutic effect in this nude mice xenograft whereas desoxyepothiloneB achieved a full cure.

[0571] Prolonged (6 hr.) i.v. infusion allowed higher doses (e.g., 30mg/kg, Q2Dx5) to be administered (without lethality) than i.v. bolusinjection of desoxyepothilone B, and yet reduced drug toxicity.

[0572] Accordingly, the present inventors have found desoxyepothilone Bto have excellent properties for therapeutic application as an MDR agentand moreover as a general anticancer agent. TABLE 9 Therapeutic Effectof Desoxyepothilone B, Taxol ®, and Adriamycin in Nude Mice BearingHuman MX-1 Xenograft^(a) Tumor Dose Average Body Weight Change (g)Average Tumor Volume (T/C) Disap- #Mice Drug (mg/kg) Day 8 10 12 14 1618 Day 10 12 14 16 18 pearance Died Control  0 24.6 −0.1 +1.0 +1.0 +1.3+1.8 1.00 1.00 1.00 1.00 1.00 0/10 0/10 Desoxyepothilone B 35 23.0 −0.1+0.7 −0.3 −1.7 −1.6 0.42 0.28 0.07 0.04 0.02 0/10 3/10 Taxol ®  5 24.0−1.3 −0.8 −1.4 −1.9 −1.8 0.58 0.36 0.34 0.42 0.47 2/10 0/10 10 24.3 −1.0−1.0 −2.3 −3.5 −3.8 0.85 0.40 0.21 0.20 0.12 2/6  1/6  Adriamycin  2^(b) 23.9 +0.3 0 −1.4 −1.9 −2.0 0.94 0.88 1.05 0.69 0.72 1/10 0/10  3^(C) 22.4 +1.3 −0.2 −1.5 −2.1 −2.3 0.72 0.54 0.56 0.51 0.36 4/6  0/6 

[0573] TABLE 10 Extended Experiment of Desoxyepothilone B, Taxol ®,Cisplatin and Cyclophosphamide in Nude Mice Bearing Human MX-1Xenograft^(a) Average Tumor Dose Average Body Weight Change (g) TumorDisappearance Disappearance # Drug (mg/kg) Day 8 20 22 24 26 28 Day 2022 24 26 28 Duration (Day) Died Desoxyepo B 40 23.0 −1.7 −2.4 −2.4 −1.4−1.2 2/10^(b) 2/10 3/10 5/10 5/10 44 (5/10) 0/10 Taxol ® 5 24.0 −1.6−0.3 +0.1 −0.6 −0.4 0/10 0/10 0/10 0/10 0/10 2/10 10 No extended test1/6 on day 16 Reappeared 2/6  on day 38

[0574] TABLE 11 Toxicity of Epothilone B and Desoxyepothilone B innormal nude mice. Dose Group and Schedule Disap- Duration (mg/kg) Numberof mice Died pearance Control 4 0 Epothilone B^(a) 0.6 QD × 4 8 8Desoxyepothilone B  25 QD × 4 6 0

[0575] TABLE 12 Therapeutic Effect of Epothilone B, Desoxyepothilone Band Taxol ® in B6D2F₁ Mice Bearing B16 Melanoma^(a) Dose Average WeightChange (g) Average Tumor Volume (T/C) # Mice Drug (mg/kg) Day 0 3 5 7 911 Day 5 7 9 11 Died Control 0 26.5 −0.2 0 −0.2 0 +1.0 1.00 1.00 1.001.00  0/15 Epothilone B 0.4 QD×6^(b) 27.1 −0.2 −0.6 −1.1 −3.4 −3.9 1.081.07 1.27 1.07 1/8 0.8 QD×5^(c) 27.0 0 −0.8 −3.1 −4.7 −4.7 0.57 0.890.46 0.21 5/8 Desoxyepothilone B  10 QD×8 27.0 −0.7 −0.9 −1.1 −1.5 -0.30.23 0.22 0.51 0.28 0/6  20 QD1-4,7-8 26.9 −1.3 −2.2 −1.3 −1.6 -0.8 0.590.63 0.58 0.33 0/6 Taxol ®   4 QD×8 26.7 +0.1 +0.2 +0.3 +0.4 +0.8 0.620.39 0.56 0.51 0/8 6.5 QD×8 26.7 +0.1 +0.3 +0.3 +0.4 +1.7 0.19 0.43 0.200.54 0/8

[0576] TABLE 13 Therapeutic Effect of Desoxyepothilone B, Epothilone B,Taxol ® and Vinblastine in Nude Mice Bearing Human MX-1 Xenograft^(a).Dose Average Body Weight Change (g) Average Tumor Volume (T/C) Drug(mg/kg) Day 7 11 13 15 17 Day 11 13 15 17 Note Control 27.9 +0.8 +1.1+1.9 +0.6 1.00 1.00 1.00 1.00 0/8 died Desoxyepothilone B 15 27.1 +0.8+1.1 +1.6 +1.5 0.65 0.46 0.49 0.41 0/6 died 25^(b) 27.0 +0.4 +0.7 +1.0+0.7 0.38 0.11 0.05 0.04 0/6 died (1/6 cured on day 35) Epothilone B 0.3 26.9 +0.5 +0.4 −0.3 −1.2 1.00 0.71 0.71 0.84 0/7 died  0.6^(c) 27.4−0.3 −1.3 −2.1 −2.1 1.08 0.73 0.81 0.74 3/7 died Taxol ®  5 26.9 −0.1+0.4 +1.1 +1.2 0.54 0.46 0.40 0.45 0/7 died 10^(d) 27.6 −2.7 −1.1 −0.3+2.2 0.43 0.37 0.12 0.11 4/7 died Vinblastine  0.2 25.7 +0.6 +1.4 +2.3+2.9 0.65 0.54 0.56 0.88 0/7 died  0.4^(c) 26.4 +0.8 +0.5 +1.9 +2.1 0.800.56 0.83 0.88 1/7 died

[0577] TABLE 14 Toxicity of Hematology and Chemistry of DesoxyepothiloneB, and Taxol ® in Nude Mice Bearing Human MX-1 Xenograft^(a)Hematology^(b) WBC Chemistry^(b) Dose Total Neutrophils Lymph RBC PLTGOT GPT Drug (mg/kg ip) (10³/mm³) (%) (%) (10³/mm³) (10⁶/mm³) (U/L)(U/L) Control 12.9 38 61 8.1  800 203 45 (n = 4) (n = 4)Desoxyepothilone B 25 and 35^(c) 11.8 48 48 8.4  700 296 55 (n = 3) (n =6) Taxol ® 5 and 6^(d) 10.9 51 48 6.1 1083 438 79 (n = 5) (n = 5) Normalrange^(c) 6.91˜12.9 8.25˜40.8 62˜90 10.2˜12.0 190˜340 260 138.7

[0578] TABLE 15 Therapeutic Effect of Desoxyepothilone B, Taxol ®,Adriamycin, and Camptothecin in Nude Mice Bearing MDR Human MCF-7/AdrTumor Dose Average Body Weight Change (g) Average Tumor Volume (T/C)Drug (mg/kg) Day 8 11 13 15 17 Day 11 13 15 17 Died Control 0 25.0 +2.0+2.6 +3.1 +3.7 1.00 1.00 1.00 1.00 0/8 DesoxyEpoB 35 25.0 +0.3 +0.7 +0.6+0.8 0.31 0.27 0.30 0.34 0/8 Taxol ® 6 25.3 +1.7 +1.8 +0.8 +0.9 0.570.66 085 0.90 0/8 12 24.5 +0.7 −1.3 −2.4 0 0.50 0.51 0.32 0.40 3/6Adriamycin 1.8 25.6 +0.2 −0.4 −0.6 −0.4 0.70 0.68 0.84 0.78 0/8 3 24.6+0.5 −1.5 −3.2 −1.6 0.66 0.83 0.57 0.53 3/6 camptothecin 1.5 24.4 +1.1+0.9 +1.7 +1.4 1.08 0.72 0.61 0.72 0/8 3.0 24.5 −0.6 −0.4 −0.8 −0.9 0.950.76 0.61 0.43 0/6

[0579] TABLE 16 Extended Experiment of Desoxyepothilone B, Taxol ® inNude Mice Bearing Human MX-1 Xenograft^(a) Average Tumor Dose AverageBody Weight Change (g) Tumor Disappearance Disappearance Drug (mg/kg)Day 8 20 22 24 26 28 Day 20 22 24 26 28 Duration (Day) Died Desoxyepo B40 23.0 −1.7 −2.4 −2.4 −1.4 −1.2 2.10^(b) 2/10 3/10 5/10 5/10 44 (5/10)0/10 Taxol ® 5 24.0 −1.6 −0.3 +0.1 −0.6 −0.4 0/10 0/10 0/10 0/10 0/102/10 10 No Extended Test 1/6 on day 16, Reappear on 2/6_((0/6)) day 38

[0580] TABLE 17 Therapeutic Effects of Desoxyepothilone B, Taxol ® inNude Mice Bearing MX-1 Xenograft. # Died of toxicity CONTROL 0/10Treatment Schedule Day 8 10 12 14 16 18 20 Tumor Size 19 ± 78 ± 151 ±372 ± 739 ± 1257 ± 1991 ± Sacrificed mm³) 2 8 15 55 123 184 331 (n = 10)DESOXYEPO- 0/10 THILONE B Dose 35 mg/kg on day 40 mg/kg on day NoTreatment Schedule Day 8 10 12 14 16 18 20 22 24 26 28 30 45 47 50 60Tumor Size Mouse 1 15 15 40 40 15 32 30 30 30 30 0 0 0 24   S* — Mouse 223 23 15 15 15 15 30 48 48 0 30 48 900 1200 S — Mouse 3 15 60 90 105 105126 96 150 180 0 48 64 600 600 S — Mouse 4 21 38 38 0 0 10 8 8 8 8 0 0 00 0 0 Mouse 5 12 23 50 12 0 4 0 0 0 0 0 0 0 0 0 0 Mouse 6 15 40 32 8 8 88 12 12 12 12 30 120 120 S — Mouse 7 21 30 15 15 8 8 8 8 8 8 8 8 180 280S — Mouse 8 20 48 70 15 15 8 8 0 0 0 0 0 0 8 S — Mouse 9 25 50 40 15 8 00 0 0 0 0 0 0 0 4 4 Mouse 10 20 38 38 38 38 25 25 25 0 0 15 15 100 100 S— Taxol ® 2/10 Dose 5 mg/kg on day 5 mg/kg on day Schedule Day 8 10 1214 16 18 20 22 24 26 28 30 45 47 50 60 Tumor Size 17 ± 45 ± 54 ± 128 ±311 ± 596 ± 1114 ± 1930 ± 2285 ± S (n = 10) 2 7 13 42 115 151 346 569597 Extended studies → Extended → Experiment ended observations

[0581] TABLE 18 Toxicity of Epothilone B and Desoxyepothilone B innormal nude mice Dose and Schedule Group (mg/kg) Number of mice DiedControl 4 0 Epothilone B^(a) 0.6 QD × 4 8 8 Desoxyepothilone B 25 QD × 46 0

[0582] TABLE 19 Therapuetic effects of desoxyepothilone B (dEpo B) andTaxol ® in nude mice bearing MX-1 xenograft^(a) Route^(b) Tumor Dosei.v. Average Body Weight (g) Average Tumor Size (T/C) Disap- Drugs(mg/kg) infusion Day 8 14 16 18 20 Day 14 16 18 20 pearance Control 030.2 +0.8 +1.7 +2.6 +3.2 1.00 1.00 1.00 1.00 0/5 Epo B 30 Q2D×5 30.3−2.7 −4.0 −4.5 −6.8 0.19 0.10 0.03  TD^(C) 3/3 axol ® 15 Q2D×5 30.8 0−1.1 −1.6 −1.4 0.06 0.01 TD TD 4/4 24 Q2D×5 28.5 −4.8 −5.3 −6.0 −6.20.03 TD TD TD 4/4

[0583] TABLE 20 Therapuetic effects of desoxyepoB (dEpo B) and Taxol ®in nude mice bearing MCF-7 Adr xenograft^(a) Route^(b) Average TumorDose i.v. Average Body Weight (g) Size (T/C) Toxicity Drug (mg/kg)infusion Day 8 14 16 18 Day 14 16 18 death Control 0 30.2 +0.8 +1.7 +2.61.00 1.00 1.00 0/5 dEpo B 30 Q2D×5 30.3 −2.7 −4.0 −4.5 0.16*** 0.150.13*** 0/3 Taxol ® 15 Q2D×5 30.8 0 −1.1 −1.6 0.81 0.89 0.76 0/4 24Q2D×5 28.5 −4.8 −5.3 −6.0 0.73 0.71* 0.73* 0/4

[0584] In further tests, desoxyepothilone B showed similar anticancerefficacy as Taxol® in regular human tumor xenographs in nude mice,represented in Table 21. However, in drug-resistant tumors,desoxyepothilone B is by far better cancer therapeutic agent whencompared with Taxol® in all tumors tested. Desoxyepothilone B is notonly superior to Taxol® in many respects but is also a bettertherapeutic agent than epothilone B, camptothecin, vinblastine,adriamycin or VP-16 (etoposide) against many other tumors. See Table 21.

[0585] Stability of Desoxyepothilone B in Plasma

[0586] As shown in FIG. 75, desoxyepothilone B is surprisingly andunexpectedly significantly more stable in human serum plasma than inmouse plasma. Desoxyepothilone B is relatively unstable in mouse plasmawith a short half-life of about 15 or 20 min, but is very stable inhuman plasma with a half-life of about 75 hours. It is therefore likelythat desoxyepothilone B can be particularly favorable in treatingpatients in view of the long lasting effects and the absence of a needto use prolonged i.v. infusions. TABLE 20A Therapuetic effects ofdesoxyepoB (dEpo B) and Taxol ® in nude mice bearing human ovarian UL3-Ctumor.^(a) Dose Average Body Weight (g) Average Tumor Size (T/C) Drug(mg/kg) Day 19 27 29 31 33 Day 27 29 31 33 Control 0 30.9 +0.9 +0.8 +0.4+0.1 1.00 1.00 1.00 1.00 dEpo B 30 30.0 −1.5 −4.0 −3.6 −2.7 0.19 0.130.03 0.03 Taxol ® 20 31.3 −2.9 −3.3 −2.4 −2.7 0.10 0.08 0.03 0.04

[0587] TABLE 21 Comparison Between dEpoB and Taxol ® Chemotherapy inNude Mice Lowest Average Therapeutic Effects Doses Tumor Size TumordEpoB vs Tumor Drugs Schedule (T/C) Disappearance Taxol ® A549 dEpoB 40mg/kg Q2D×3 0.01 1/3 ≅ Lung Taxol ® 15 mg/kg Q2D×3 0.01 2/4 MX-1 dEpoB30 mg/kg Q2D×6 0 5/5 ≅ Taxol ® 15 mg/kg Q2D×6 0 5/5 HT-29 dEpoB 30 mg/kgQ2D×6 0.02 0 ≦ Colon Taxol ® 15 mg/kg Q2D×6 0.01 0 UL3-C dEpoB 30 mg/kgQ2D×5 0.03 0 ≧ Ovary Taxol ® 15 mg/kg Q2D×5 0.04 0 PC-3 dEpoB 40 mg/kgQ2D×3 0.12 0 << Prostate Taxol ® 15 mg/kg Q2D×3 0.02 0 SK-OV-3 dEpoB 30mg/kg Q2D×6 0.17 0 << Ovary Taxol ® 15 mg/kg Q2D×6 0.02 1/4 MCF-7/AdrdEpoB 30 mg/kg Q2D×5 0.11 0 >> Breast Taxol ® 24 mg/kg Q2D×5 0.71 0CCRF/Taxol dEpoB 30 mg/kg Q2D×6 0 3/3 >>> Leukemia Taxol ® 15 mg/kgQ2D×6 1.09 0 CCRF/VBL dEpoB 30 mg/kg Q2D×5 0 2/2 >>> Leukemia Taxol ® 15mg/kg Q2D×5 0.76 0 CCRF/CEM dEpoB 30 mg/kg Q2D×5 0 2/2 ≅ LeukemiaTaxol ® 15 mg/kg Q2D×5 0 2/2

[0588] TABLE 22 Comparison of Cancer Chemotherapeutic Effects Route ofTherapeutic effect Tumor Administation Rank Order B16 Melanoma i.p.dEpoB > Taxol ® > > EpoB MX-1 i.p. dEpoB > Camptothecin > Taxol ® > VBL,EpoB MX-1 i.p. dEpoB > Adriamycin > Taxol ® SK-OV-3 i.v./i.p. Taxol ® ≈dEpoB PC-3 i.v./i.p. Taxol ® > dEpoB > > Adriamycin MCF-7/Adr i.p.dEpoB > > Camptothecin > Adriamycin > Taxol ® MX-1 i.v. infusion Taxol ®≈ dEpoB MCF-7/Adr i.v. infusion dEpoB > > VP-16, Taxol ® > VBL,Adriamycin CCRF-CEM i.v. infusion dEpoB ≈ Taxol ® CCRF-CEM/Taxol ® i.v.infusion dEpoB > > > Taxol ® CCRF-CEM/VBL i.v. infusion dEpoB > > >Taxol ® SK-OV-3 i.v. infusion Taxol ® > dEpoB HT-29 i.v. infusionTaxol ® ≧ dEpoB A549 i.v. infusion Taxol ®, dEpoB, VBL > VP-16 PC-3 i.v.infusion Taxol ®, VBL ≧ dEpoB > > VP-16

[0589] TABLE 23 Therapeutic Effects of DesoxyepoB and Taxol ® in NudeMice Bearing Human Lymphoblastic Leukemia CCRF-CEM^(a). Average BodyWeight Changes (g) Drug Dose Day 21 23 25 27 29 31 33 35 37 39 41Control 0 29.0 +0.1 −1.1 −0.2 +0.8 +0.1 End End End End End DesoxyepoB40 26.6 −1.4 −3.6 −3.9 −5.2 −4.2 −3.1 −2.3 −1.2 −0.2 +0.9 Taxol ® 2029.0 −0.1 −1.9 −2.9 −3.0 −3.6 −2.6 −0.5 +1.2 +1.3 +2.1 Average TumorSize (T/C) Proportion of Tumor Disappearance (n/total) Day 21 23 25 2729 31 33 35 37 39 41 Control  0 1.00 1.00 1.00 1.00 1.00 End End End EndEnd End 0/5 0/5 0/5 0/5 0/5 DesoxyepoB 40 1.10 0.73 0.31 0.09 0 0 0 0 ND^(b) ND ND 0/3 0/3 0/3 0/3 3/3 3/3 3/3 3/3 1/3 1/3 0/3 Taxol ® 201.17 0.78 0.44 0.11 0 0 0 0 ND ND ND 0/4 0/4 0/4 1/4 4/4 4/4 4/4 1/4 0/40/4 0.4

[0590] TABLE 24 Therapeutic Effects of dEpoB, Taxol ®, VBL and VP-16 inNude Mice Bearing Human Lung Carcinoma A549 Dose i.v. Tumor (mg/kg)infusion Average Body Weight (g) Average Tumor Size (mm³) Disap- DrugsQ2D × 3 (hrs) Day 7 11 13 15 Day 11 13 15 17 pearance Control 0 6 26.4+1.3 +0.6 +0.2 96 128 306 553 0/4 dEpoB 30 18 29.3 −2.0 −5.8 −5.6 17 2126 21 1/3 40 6 27.8 −1.6 −3.2 −2.4 31 19 4 4 1/3 50 6 27.1 −2.0 −3.2−2.5 33 19 6 6 1/2 Taxol ® 15 6 27.6 −1.2 −0.8 +0.5 49 32 7 6 2/4 24 628.5 −2.1 −2.7 −0.9 14 8 0 0 4/4 VBL 2 i.v. inj 25.5 +13.4 +2.3 +3.2 1111 11 7 2/3 VP-16 25 i.v. inj 27.2 +0.1 −0.2 −2.2 59 150 275 536 0/3

[0591] TABLE 25 Therapeutic Effects of dEpoB, Taxol ®, VBL and VP-16 inNude Mice Bearing Human Prostate Adenocarcinoma PC-3 i.v. Average TumorTumor Dose infusion Average Body Weight (g) Size (TC) Disap- Drugs(mg/kg) (hrs) Day 5 9 11 13 Day 9 11 13 pearance Control 0 6 26.5 +1.4+0.5 +0.1 1.00 1.00 1.00 0/5 dEpoB 30 18 29.4 −2.2 −5.9 −5.8 0.17 0.060.06 0/3 40 6 28.3 −1.7 −3.4 −2.7 0.40 0.19 0.12 0/3 50 6 28.1 −2.1 −3.0−2.6 0.37 0.13 0.03 0/3 Taxol ® 16 6 26.6 −1.1 −0.9 +0.4 0.34 0.09 0.020/4 24 6 28.8 −2.2 −2.8 −0.8 0.18 0.05 0.06 2/4 VBL 2 i.v. inj 23.5 +1.4+2.1 +3.0 0.37 0.13 0.03 1/3 VP-16 25 i.v. inj 24.9 +0.3 −0.1 −2.4 1.001.12 0.88 0/3

[0592] TABLE 26 Therapeutic Effects of DesoxyepoB and Taxol ® in NudeMice Bearing Human Lymphoblastic Leukemia CCRF-CEM. Tumor Average BodyWeight Change (g) Average Tumor Size (T/C) Disap- Drug Q2D × 4 Dose Day21 23 25 27 29 31 Day 23 25 27 29 31 pearance Control 0 29.0 +0.1 −1.1−0.2 +0.8 +0.1 1.00 1.00 1.00 1.00 1.00 0/5 DesoxyepoB 40 26.6 −1.4 −3.6−3.9 −5.2 −4.2 0.68 0.30 0.09 0 0 3/3 Taxol 20 29.0 −0.1 −1.9 −2.9 −3.0−3.6 0.78 0.44 0.11 0 0 4/4

[0593] TABLE 27 Comparison of Therapeutic Effects and Toxicity of dEpoBand Paclitaxel with Different Solvents, Routes and Schedules ofAdministration Using a Non-MDR MX-1 Xenograft^(a) Therapeutic EffectToxicity toward Dose against MX-1 Xenograft^(d) Nude Mice^(e) RouteSolvent^(b) (mg/kg)^(c) dEpoB Paclitaxel DepoB Paclitaxel i.p. DMSO 35Q2D×5 +++++ +++++ ++ ++ Cremophor- 15 Q2D×5 + ++ +++++ ++++ EtOH i.v.DMSO 15 Q2D×5 ++ ++++ +++ ++ injection 24 Q2D×5 ++ ++++ +++ ++ i.v.Cremophor- 30 Q2D×5 +++++ +++++ ++ ++ infusion EtOH 60 Q4D×2 +++++ND^(f) +++ ND^(f) 6 hr 24 hr

What is claimed is:
 1. A method of preparing a desoxyepothilone having the structure:

wherein R, R₀, and R′ are independently H, linear or branched chain alkyl, optionally substituted by hydroxy, alkoxy, carboxy, carboxaldehyde, linear or branched alkyl or cyclic acetal, fluorine, NR₁R₂, N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ are independently H, phenyl, benzyl, linear or branched chain alkyl; wherein R″ is —CY═CX—, or H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein X is H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein Z is O, N(OR₃) or N—NR₄R₅, wherein R₃, R₄ and R₅ are independently H or a linear or branched alkyl; and wherein n is 0, 1, 2, or 3; which comprises treating an epothilone having a structure:

wherein R, R₀, R₁, R₂, R₃, R₄, R₅, R′, R″, X, Y, Z and n are defined as for the desoxyepothilone, under suitable conditions so as to deoxygenate the epothilone, and thereby to provide the desoxyepothilone.
 2. The method of claim 1 wherein desoxyepothilone has the structure:

wherein R is H, methyl, ethyl, n-propyl, n-butyl, n-hexyl,

or (CH₂)₃—OH.
 3. The method of claim 1 wherein the epothilone is deoxygenated using a zinc/copper couple.
 4. The method of claim 1 wherein the epothilone is deoxygenated in the presence of a polar solvent comprising isopropanol and water.
 5. A method of preparing a desoxyepothilone having the structure:

wherein R, R₀, and R′ are independently H, linear or branched chain alkyl, optionally substituted by hydroxy, alkoxy, carboxy, carboxaldedyde, linear or branched alkyl or cyclic acetal, fluorine, NR₁R₂, N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ are independently H, phenyl, benzyl, linear or branched chain alkyl; wherein R″ is —CY═CHX, or H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein X is H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein Z is O, N(OR₃) or N—NR₄R₅, wherein R₃, R₄ and R₅ are independently H or a linear or branched chain alkyl; and wherein n is 0, 1, 2, or 3; which comprises treating an epothilone having a structure:

wherein R, R₀, R₁, R₂, R₃, R₄, R₅, R′, R″, X, Y, Z and n are defined as for the desoxyepothilone, under suitable conditions so as to deoxygenate the epothilone, and thereby to provide the desoxyepothilone.
 6. The method of claim 5 wherein the desoxyepothilone has the structure:

wherein R is H, methyl, ethyl, n-propyl, n-butyl, n-hexyl or hydroxypropyl.
 7. The method of claim 5 wherein the epothilone is deoxygenated using a zinc/copper couple.
 8. The method of claim 5 wherein the epothilone is deoxygenated in the presence of a polar solvent comprising isopropanol and water.
 9. A method of preparing a desoxyepothilone having the structure:

wherein R, R₀, and R′ are independently H, linear or branched chain alkyl, optionally substituted by hydroxy, alkoxy, carboxy, carboxaldedyde, linear or branched alkyl or cyclic acetal, fluorine, NR₁R₂, N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ are independently H, phenyl, benzyl, linear or branched chain alkyl; wherein R″ is —CY═CHX, or H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein X is H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein Y is H or linear or branched chain alkyl; and wherein Z is O, N(OR₃) or N—NR₄R₅ where R₃, R₄ and R₅ are independently H or a linear or branched alkyl; and wherein n is 0, 1, 2, or 3; which comprises treating a protected desoxyepothilone having the structure:

wherein R_(A) is a linear or branched alkyl, alkoxyalkyl, substituted or unsubstituted aryloxyalkyl, trialkylsilyl, aryldialkylsilyl, diarylalkylsilyl, triarylsilyl, linear or branched acyl, substituted or unsubstituted aroyl or benzoyl; and wherein R_(B) is hydrogen, t-butyloxycarbonyl, amyloxycarbonyl, (trialkylsilyl)alkyloxycarbonyl, (dialkylarylsilyl) alkyloxycarbonyl, benzyl, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, a linear or branched acyl, substituted or unsubstituted aroyl or benzoyl; under suitable conditions to form the desoxyepothilone.
 10. The method of claim 9 wherein n is 3 and R″ is 2-methyl-1,3-thiazolinyl.
 11. The method of claim 9 wherein R_(A) is TES and R_(B) is Troc.
 12. The method of claim 9 wherein the treating step comprises contacting the protected desoxyepothilone (i) with SmX₂, where X is Cl, Br or 1, in the presence of a polar nonaqueous solvent selected from the group consisting of tetrahydrofuran, p-dioxane, diethyl ether, acetonitrile and N,N-dimethylformamide, and optionally in the presence of N,N-dimethyl-N′-propylurea or hexamethylphosphoramide and (ii) with a source of fluoride ion selected from the group consisting of tetra-n-methylammonium fluoride, tetra-n-butylammonium fluoride and HF.pyridine.
 13. A method of preparing a protected desoxyepothilone having the structure:

wherein R, R₀, and R′ are independently H, linear or branched chain alkyl, optionally substituted by hydroxy, alkoxy, carboxy, carboxaldehyde, linear or branched alkyl or cyclic acetal, fluorine, NR₁R₂, N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ are independently H, phenyl, benzyl, linear or branched chain alkyl; wherein R″ is —CY═CHX, or H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein X is H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein n is 2 or 3; wherein R_(A) is a linear or branched alkyl, alkoxyalkyl, substituted or unsubstituted aryloxyalkyl, trialkylsilyl, aryldialkylsilyl, diarylalkylsilyl, triarylsilyl, linear or branched acyl, substituted or unsubstituted aroyl or benzoyl; and wherein R_(B) is hydrogen, t-butyloxycarbonyl, amyloxycarbonyl, (trialkylsilyl)alkyloxycarbonyl, (dialkylarylsilyl)alkyloxycarbonyl, benzyl, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, a linear or branched acyl, substituted or unsubstituted aroyl or benzoyl; which comprises cyclocondensing a hydroxy acid desoxyepothilone precursor having the structure:

wherein R, R₀, R_(A), R_(B), R′, R″ and n are defined as above; under suitable conditions to form the protected desoxyepothilone.
 14. The method of claim 13 wherein n is 3 and R″ is 2-methyl-1,3-thiazolinyl.
 15. The method of claim 13 wherein R_(A) is TES and R_(B) is Troc.
 16. The method of claim 13 wherein the hydroxy acid desoxyepothilone precursor is cyclocondensed using a cyclocondensing reagent selected from the group consisting of acetic anhydride, pentafluorophenol, 2,4-dichlorobenzoyl chloride and 2,4,6-trichlorobenzoyl chloride.
 17. The method of claim 13 wherein the hydroxyacid is cyclocondensed using 2,4,6-trichlorobenzoyl chloride in the presence of a tertiary amine selected from the group consisting of triethyl amine, tri-n-propylamine, diisopropylethylamine and diethyliso-propylamine, and optionally in the presence of pyridine or N,N-dimethylaminopyridine.
 18. A method of preparing a hydroxy acid desoxyepothilone precursor having the structure:

wherein R, R₀, and R′ are independently H, linear or branched chain alkyl, optionally substituted by hydroxy, alkoxy, carboxy, carboxaldehyde, linear or branched alkyl or cyclic acetal, fluorine, NR₁R₂, N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ are independently H, phenyl, benzyl, linear or branched chain alkyl; wherein R″ is —CY═CHX, or H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein X is H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein n is 2 or 3; wherein R_(A) is a linear or branched alkyl, alkoxyalkyl, substituted or unsubstituted aryloxyalkyl, trialkylsilyl, aryidialkylsilyl, diarylalkylsilyl, triarylsilyl, linear or branched acyl, substituted or unsubstituted aroyl or benzoyl; wherein R_(B) is hydrogen, t-butyloxycarbonyl, amyloxycarbonyl, (trialkylsilyl) alkyloxycarbonyl, (dialkylarylsilyl)alkyloxycarbonyl, benzyl, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, linear or branched acyl, substituted or unsubstituted aroyl or benzoyl; which comprises selectively etherifying and hydrolyzing a hydroxy ester desoxyepothilone precursor having the structure:

wherein R, R₀, R_(B), R_(C), R′, R″ and n are defined as above; and wherein R_(C) is tertiary-alkyl; under suitable conditions to form the hydroxy acid desoxyepothilone precursor.
 19. The method of claim 18 wherein n is 3 and R″ is 2-methyl-1,3-thiazolinyl.
 20. The method of claim 18 wherein R_(A) is TES and R_(B) is Troc.
 21. The method of claim 18 wherein the selective etherifying step comprises contacting the hydroxy ester desoxyepothilone precursor with a silylating reagent to form an ether intermediate, and the hydrolyzing step comprises contacting the ether intermediate with a protic acid or tetra-n-butylammonium fluoride.
 22. The method of claim 21 wherein the silylating reagent is TESOTf in the presence of 2,6-lutidine.
 23. The method of claim 21 wherein the protic acid is HCl in the presence of methyl alcohol.
 24. A method of preparing a hydroxy ester desoxyepothilone precursor having the structure:

wherein R, R₀, and R′ are independently H, linear or branched chain alkyl, optionally substituted by hydroxy, alkoxy, carboxy, carboxaldehyde, linear or branched alkyl or cyclic acetal, fluorine, NR₁R₂, N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ are independently H, phenyl, benzyl, linear or branched chain alkyl; wherein R″ is —CY═CHX, or H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein X is H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein n is 2 or 3; wherein R_(B) is hydrogen, t-butyloxycarbonyl, amyloxycarbonyl, (trialkylsilyl)alkyloxycarbonyl, (dialkylarylsilyl)alkyloxycarbonyl, benzyl, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, linear or branched acyl, substituted or unsubstituted aroyl or benzoyl; and wherein R_(C) is tertiary-alkyl; which comprises reducing a hydroxy ketoester desoxyepothilone precursor having the structure:

wherein P, R, R₀, R_(B), R_(C), R′, R″ and n are defined as above; under suitable conditions to form the hydroxy ester desoxyepothilone precursor.
 25. The method of claim 24 wherein n is 3 and R″ is 2-methyl-1,3-thiazolinyl.
 26. The method of claim 24 wherein R_(A) is TES and R_(B) is Troc.
 27. The method of claim 24 wherein the reducing step comprises contacting the hydroxy ketoester desoxyepothilone precursor with a stereospecific reducing reagent.
 28. The method of claim 24 wherein the stereospecific reducing reagent comprises hydrogen gas at from about 900 pounds per square inch to about 2200 pounds per square inch in the presence of (R)-(BINAP)RuCl₂ and optionally in the presence of HCl and an alcohol selected from the group consisting of MeOH, EtOH, and i-PrOH.
 29. A method of preparing a hydroxy ketoester desoxyepothilone precursor having the structure:

wherein P is H; wherein R, R₀, and R′ are independently H, linear or branched chain alkyl, optionally substituted by hydroxy, alkoxy, carboxy, carboxaldehyde, linear or branched alkyl or cyclic acetal, fluorine, NR₁R₂, N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ are independently H, phenyl, benzyl, linear or branched chain alkyl; wherein R″ is —CY═CHX, or H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein X is H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein n is 2 or 3; wherein R_(B) is hydrogen, t-butyloxycarbonyl, amyloxycarbonyl, (trialkylsilyl)alkyloxycarbonyl, (dialkylarylsilyl) alkyloxycarbonyl, benzyl, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, a linear or branched acyl, substituted or unsubstituted aroyl or benzoyl; and wherein R_(C) is tertiary-alkyl; which comprises deprotecting a protected ketoester desoxyepothilone precursor having the structure:

wherein R, R₀, R_(A), R_(B), R_(C), R′, R″ and n are defined as above; and wherein P is a linear or branched alkyl, alkoxyalkyl, substituted or unsubstituted aryloxyalkyl, trialkylsilyl, aryldialkylsilyl, diarylalkylsilyl or triarylsilyl; under suitable conditions to form the hydroxy ketoester desoxyepothilone precursor.
 30. The method of claim 29 wherein n is 3 and R″ is 2-methyl-1,3-thiazolinyl.
 31. The method of claim 29 wherein R_(A) is TES and R_(B) is Troc.
 32. The method of claim 29 wherein P is TBS.
 33. The method of claim 29 wherein the deprotecting step comprises contacting the protected ketoester desoxyepothilone precursor with a protic acid.
 34. The method of claim 33 wherein the protic acid is HCl in methyl alcohol or ethyl alcohol.
 35. A method of preparing a protected ketoester desoxyepothilone precursor having the structure:

wherein P is a linear or branched alkyl, alkoxyalkyl, substituted or unsubstituted aryloxyalkyl, trialkylsilyl, aryldialkylsilyl, diarylalkylsilyl or triarylsilyl; wherein R, Ro, and R′ are independently H, linear or branched chain alkyl, optionally substituted by hydroxy, alkoxy, carboxy, carboxaldehyde, linear or branched alkyl or cyclic acetal, fluorine, NR₁R₂, N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ are independently H, phenyl, benzyl, linear or branched chain alkyl; wherein R″ is —CY═CHX, or H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein X is H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein n is 2 or 3; wherein R_(B) is hydrogen, t-butyloxycarbonyl, amyloxycarbonyl, (trialkylsilyl)alkyl-oxycarbonyl, (dialkylarylsilyl)alkyloxycarbonyl, benzyl, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, a linear or branched acyl, substituted or unsubstituted aroyl or benzoyl; and wherein R_(C) is tertiary-alkyl; which comprises coupling a terminal vinyl enol ether ester having the structure:

wherein R, R₀, R_(B), R_(C), and R′ are defined as above; wherein m is 0, 1 or 2; and wherein R_(D) is linear or branched alkyl, benzyl, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, linear or branched acyl, substituted or unsubstituted aroyl or benzoyl; with a protected halovinyl or metalvinyl compound having the structure:

wherein R, P and R″ are defined as above; and wherein Q is a halide or a metal; under suitable conditions to form the protected ketoester desoxyepothilone precursor.
 36. The method of claim 35 wherein n is 3 and R″ is 2-methyl-1,3-thiazolinyl.
 37. The method of claim 35 wherein R_(A) is TES and R_(B) is Troc.
 38. The method of claim 35 wherein P is TBS or TES.
 39. The method of claim 35 wherein Q is iodine or bromine.
 40. The method of claim 35 wherein R_(D) is methyl or TES.
 41. The method of claim 35 wherein the coupling step comprises contacting the terminal vinyl enol ether ester and the protected halovinyl compound with noble metal complex capable of effecting a Suzuki coupling.
 42. The method of claim 35 wherein the noble metal complex is Pd(dppf)₂Cl₂ in the presence of Ph₃As and Cs₂CO₃.
 43. A method of preparing a terminal vinyl enol ether ester having the structure:

wherein R₀ and R′ are independently H, linear or branched chain alkyl, optionally substituted by hydroxy, alkoxy, carboxy, carboxaldehyde, linear or branched alkyl or cyclic acetal, fluorine, NR₁R₂, N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ are independently H, phenyl, benzyl, linear or branched chain alkyl; wherein m is 0, 1 or 2; wherein R_(B) is hydrogen, t-butyloxycarbonyl, amyloxycarbonyl, (trialkylsilyl)alkyl-oxycarbonyl, (dialkylarylsilyl)alky-loxycarbonyl, benzyl, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, linear or branched acyl, substituted or unsubstituted aroyl or benzoyl; wherein R_(C) is tertiary-alkyl; and wherein R_(D) is linear or branched alkyl, benzyl, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarysilyl, linear or branched acyl, substituted or unsubstituted aroyl or benzoyl; which comprises: (a) treating a keto enol ester having the structure:

 under suitable conditions to form an enolate enol ester having the structure:

 wherein M is Li, Na or K; and (b) coupling the enolate enol ester with a vinyl aldehyde having the structure:

wherein m, and R₀ and R′ are as defined above; under suitable conditions to form the terminal vinyl enol ether ester.
 44. The method of claim 43 wherein the treating step comprises contacting the keto enol ester with a strong nonnucleophilic base selected from the group consisting of lithium diethylamide, lithium diethylamide, lithium diisopropylamide, lithium hydride, sodium hydride, potassium hydride and potassium t-butoxide.
 45. The method of claim 44 wherein the treating step is effected in a polar nonaqueous solvent selected from the group consisting of tetrahydrofuran, diethyl ether, di-n-propyl ether and dimethylformamide at a temperature from about −100° C. to about +10° C.
 46. The method of claim 44 wherein the temperature is from about −20° C. to −40° C.
 47. The method of claim 43 wherein the coupling step comprises contacting the enolate enol ester with the vinyl aldehyde at a temperature from about −130° C. to about −78° C. 